Lymphoid tissue-specific cell production from hematopoietic progenitor cells in three-dimensional devices

ABSTRACT

The invention relates to a method for lymphoid tissue-specific cell production from hematopoietic progenitor cells in unique, three-dimensional culture devices, in the presence of lymphoreticular stromal cells and in the absence of exogenously added growth factors. The resulting differentiated progeny. The lymphoid tissue-specific cells may be isolated at any sequential stage of differentiation and further expanded. The lymphoid tissue-specific cells also may be genetically altered at any stage of the process.

RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US99/26795 applicationfiled on Nov. 12, 1999, entitled LYMPHOID TISSUE-SPECIFIC CELLPRODUCTION FROM HEMATOPOIETIC PROGENITOR CELLS IN THREE-DIMENSIONALDEVICES, from which priority under 35 U.S.C. §365(a) is claimed, andwhich in turn claims priority from US provisional application Ser. No.60/107,972 filed Nov. 12, 1998. The contents of the PCT application andthe provisional application are hereby expressly incorporated byreference.

FIELD OF THE INVENTION

The invention pertains to the co-culture of hematopoietic progenitorcells and lymphoreticular stromal cells in three-dimensional devices,resulting in unexpectedly high numbers of lymphoid tissue-specific cellprogeny.

BACKGROUND OF THE INVENTION

A characteristic of the immune system is the specific recognition ofantigens. This includes the ability to discriminate between self andnon-self antigens and a memory-like potential that enables a fast andspecific reaction to previously encountered antigens. The vertebrateimmune system reacts to foreign antigens with a cascade of molecular andcellular events that ultimately results in the humoral and cell-mediatedimmune response.

The major pathway of the immune defense involving antigen-specificrecognition commences with the trapping of the antigen by antigenpresenting cells (APCs), such as dendritic cells or macrophages, and thesubsequent migration of these cells to lymphoid organs (e.g., thymus).There, the APCs present antigen to subclasses of T cells classified asmature T helper cells. Upon specific recognition of the presentedantigen, the mature T helper cells can be triggered to become activatedT helper cells. The activated T helper cells regulate both the humoralimmune response by inducing the differentiation of mature B cells toantibody producing plasma cells and the cell-mediated immune response byactivation of mature cytotoxic T cells.

The thymus has been shown to be an obligatory factor in T celldifferentiation of hematopoietic cells. Based upon the murine model, itis believed that the presence of a three dimensional organ is required,as in vitro models that do not include the thymus and a threedimensional structure fail to support T cell lymphopoiesis (Owen J J, etal., Br Med Bull., 1989, 45:350-360). The process of differentiation,however, appears to begin prior to progenitor cells contacting thethymus.

Primitive hematopoietic progenitors in the fetal liver or bone marrowgive rise to lineage committed cells, including progenitors committed tothe T lymphoid lineage. These most immature cells are identified by thesurface expression of CD34. T cell lineage committed cells express CD34,but no discrete expression of other epitopes found only on T cellprogenitors has been described. Further, T lymphocyte differentiationnormally occurs via a series of discrete developmental stages. Primitiveprogenitor cells which do not express lymphocyte specific cell surfacemarkers (CD34+ CD3− CD4− CD8−) migrate to the thymus where they acquire,through a series of maturational events, the phenotype CD34− CD3− CD4+CD8−. These cells then mature into double positive CD4+ CD8+ cells, mostof which are CD3+, although CD3 expression is not universallydetectable. These cells further undergo both positive and negativeselection, and mature to develop into single positive T cells (CD4+ CD8−or CD4− CD8+). These cells ultimately migrate into the peripheralcirculation as naive T cells.

T cell disorders and diseases represent major health problems. Recentprogress has been made using gene therapy to treat conditions involvingT lymphocytes, including AIDS. This has fostered increased interest inthe development of laboratory techniques that allow in vitro evaluationsof potential genetic therapies for these conditions.

The understanding of T cell differentiation has been hampered by thelimited availability of technologies which permit in vitro T celldifferentiation. To date, T cell differentiation studies have beenlargely confined to the SCID-hu mouse in vivo model. In vitrotechnologies have been based on thymic explant studies and primatethymic monolayers. In a recent advance, primate thymic stroma cultureshave been shown to provide an expedient, although inefficient, systemfor examining T cell development, enabling in vitro T celldifferentiation in a reproducible manner. However, the purity and numberof T cells generated this way, as well as the relatively short half-lifeof the cultures, generally results in limited applicability to moreadvanced studies of T cell differentiation and function.

SUMMARY OF THE INVENTION

The invention, in one important part, involves improved methods forculturing hematopoietic progenitor cells that direct their developmenttoward lymphoid tissue-specific lineages without the addition ofexogenous growth factors. Thus, one aspect of the invention is theculture of hematopoietic progenitor cells to generate progeny committedto a specific lineage. Another aspect is an improvement in the rate andthe number of differentiated progeny that can be obtained from a sampleof hematopoietic progenitor cells.

We describe herein a system that takes advantage of biocompatible,open-pore, three-dimensional matrices, and uses human and non-humanlymphoreticular stromal cells to provide the appropriate conditions forthe expansion and differentiation of human and non-human hematopoieticprogenitor cells toward a specific cell lineage. T lymphocytes, forexample, derived from these cultures respond normally to a variety ofstimuli and express the diversity of markers expected of mature T cells.

This system provides significant advantages over existing techniques.For example, it can provide for the rapid generation of a large numberof differentiated progeny necessary for laboratory analysis and/ortherapeutic uses, including for in vitro testing of potential genetherapy strategies or for reinfusion into subjects in vivo. The matrixitself can be implanted into subjects for in vivo studies ofhematopoietic cell growth. The system also can reasonably replicate thecomplex process of hematopoietic cell maintenance, expansion and/ordifferentiation toward a specific lineage.

Surprisingly, according to the invention, it has been discovered thathematopoietic progenitor cells co-cultured with lymphoreticular stromalcells in a porous solid scaffold, without the addition of exogenousgrowth agents, generate at a fast rate an unexpectedly high number offunctional, differentiated progeny of a lymphoid-specific lineage. Thelymphoid tissue from which lymphoreticular stromal cells are derivedhelps determine the lineage-commitment hematopoietic progenitor cellsundertake, resulting in the lineage-specificity of the differentiatedprogeny. Also surprising, according to the invention, is the discoverythat lesser amounts of nonlymphoid cells (i.e. myelo-monocytic cells)are generated from the co-culture of hematopoietic progenitor cells andlymphoreticular stromal cells in a porous solid scaffold of theinvention when compared to existing methodology. Thus, the presentinvention permits for the rapid generation of a large number ofdifferentiated, lymphoid-specific cells from a relatively small numberof hematopoietic progenitor cells. Such results were never beforerealized using known art methodologies (e.g., as in U.S. Pat. No.5,677,139 by Johnson et al., which describes the in vitrodifferentiation of CD3⁺ cells on primate thymic stroma monolayers, or asin U.S. Pat. No. 5,541,107 by Naughton et al., which describes athree-dimensional bone marrow cell and tissue culture system).

According to one aspect of the invention, a method for in vitroproduction of lymphoid tissue-specific cells is provided. The methodinvolves introducing an amount of hematopoietic progenitor cells and anamount of lymphoreticular stromal cells into a porous, solid matrixhaving interconnected pores of a pore size sufficient to permit thehematopoietic progenitor cells and the lymphoreticular stromal cells togrow throughout the matrix. The hematopoietic progenitor cells and thelymphoreticular stromal cells are then co-cultured. The amount of thelymphoreticular stromal cells utilized is sufficient to support thegrowth and differentiation of the hematopoietic progenitor cells. In oneembodiment, co-culturing occurs under conditions sufficient to produceat least a 10-fold increase in the number of lymphoid tissue-specificcells. In preferred embodiments, co-culturing occurs under conditionssufficient to produce at least a 20, 50, 100, 200, 300 or 400-foldincrease in the number of lymphoid tissue-specific cells. In someembodiments, after the co-culturing, harvesting of the lymphoidtissue-specific cells may be performed.

In certain embodiments, the hematopoietic progenitor cells may bepluripotent stem cells, multipotent progenitor cells and/or progenitorcells committed to specific hematopoietic lineages. The progenitor cellscommitted to specific hematopoietic lineages may be of T cell lineage, Bcell lineage, dendritic cell lineage, Langerhans cell lineage and/orlymphoid tissue-specific macrophage cell lineage.

The hematopoietic progenitor cells may be derived from a tissue such asbone marrow, peripheral blood (including mobilized peripheral blood),umbilical cord blood, placental blood, fetal liver, embryonic cells(including embryonic stem cells), aortal-gonadal-mesonephros derivedcells, and lymphoid soft tissue. Lymphoid soft tissue includes thethymus, spleen, liver, lymph node, skin, tonsil and Peyer's patches. Inother embodiments, the lymphoreticular stromal cells may be also derivedfrom at least one of the foregoing lymphoid soft tissues. In importantembodiments, the lymphoreticular stromal cells are thymic stromal cellsand the multipotent progenitor cells and/or committed progenitor cellsare committed to a T cell lineage. In further important embodiments, thelymphoreticular stromal cells are skin-derived stromal cells and themultipotent progenitor cells and/or committed progenitor cells arecommitted to a T cell lineage. In other embodiments, the hematopoieticprogenitor cells and/or the lymphoreticular stromal cells may begenetically altered.

In certain embodiments, the hematopoietic progenitor cells and thelymphoreticular stromal cells are autologous (e.g., originate from thesame individual). In important embodiments, the method further comprisesantigen presenting cells. In some embodiments, the hematopoieticprogenitor cells, the lymphoreticular stromal cells, and the antigenpresenting cells are all autologous. In one embodiment, the methodfurther comprises antigen presenting cells non-autologous to thehematopoietic progenitor cells and the lymphoreticular stromal cells.

In some embodiments, the hematopoietic progenitor cells and thelymphoreticular stromal cells are non-autologous (e.g., allogeneic,syngeneic and/or xenogeneic in origin). In important embodiments, themethod further comprises antigen presenting cells. Various embodimentsare provided wherein different source combinations for each of the cellsin the co-culture are encompassed by the present invention. For example,the hematopoietic progenitor cells, the lymphoreticular stromal cells,and the antigen presenting cells can be autologous, or non-autologousand each one from a different source. In another instance, thehematopoietic progenitor cells and the antigen presenting cells can beautologous or non-autologous. In still another instance, thelymphoreticular stromal cells and the antigen presenting cells arenon-autologous.

In certain embodiments, antigen presenting cells may be added to theco-culture of hematopoietic progenitor cells and lymphoreticular stromalcells. Various embodiments are provided encompassing different sourcecombinations for each of the cells in the co-culture, and wherein thelymphoid tissue-specific cells produced are to be used intransplantation into a host. For example, each of the hematopoieticprogenitor cells, lymphoreticular stromal cells and/or antigenpresenting cells may be autologous or non-autologous to the cells of thehost.

In any of the foregoing aspects and embodiments of the invention atleast one antigen may be included, or added after, the co-culture of thecells. In any of the foregoing embodiments involving antigen presentingcells, it is preferred that the antigen presenting cells are mature.

According to any of the foregoing aspects and embodiments, the method ofthe invention can include hematopoietic progenitor cells,lymphoreticular stromal cells and/or antigen presenting cells that aregenetically altered.

In one important embodiment of the invention, the hematopoieticprogenitor cells are of human origin and the lymphoreticular stromalcells are also of human origin. Antigen presenting cells can also be ofhuman and non-human origin. In another embodiment, the hematopoieticprogenitor cells are of human origin and the lymphoreticular stromalcells are of non-human origin. In preferred embodiments, non-humanlymphoreticular stromal cells are of murine origin.

In certain embodiments, the lymphoreticular stromal cells are seeded tothe matrix at the same time as the hematopoietic progenitor cells. Inother embodiments, the lymphoreticular stromal cells are seeded to thematrix prior to inoculating the hematopoietic progenitor cells.

The porous matrix can be one that is an open cell porous matrix having apercent open space of at least 50%, and preferably at least 75%. In oneembodiment the porous solid matrix has pores defined by interconnectingligaments having a diameter at midpoint, on average, of less than 150μm. Preferably the porous solid matrix is a metal-coated reticulatedopen cell foam of carbon containing material, the metal coating beingselected from the group consisting of tantalum, titanium, platinum(including other metals of the platinum group), niobium, hafnium,tungsten, and combinations thereof. In preferred embodiments, whetherthe porous solid matrix is metal-coated or not, the matrix is coatedwith a biological agent selected from the group consisting of collagens,fibronectins, laminins, integrins, angiogenic factors, anti-inflammatoryfactors, glycosaminoglycans, vitrogen, antibodies and fragments thereof,functional equivalents of these factors (including fragments thereof),and combinations thereof. Most preferably the metal coating is tantalumcoated with a biological agent. In certain other embodiments, the poroussolid matrix having seeded hematopoietic progenitor cells and theirprogeny, and lymphoreticular stromal cells (and/or antigen presentingcells), is impregnated with a gelatinous agent that occupies pores ofthe matrix.

The preferred embodiments of the invention are solid, unitarymacrostructures, i.e. not beads or packed beads. They also involvenonbiodegradable materials.

According to any of the foregoing embodiments, the method of theinvention can include culturing the cells in an environment that is freeof hematopoietic progenitor cell survival and proliferation factors suchas interleukins 3, 6 and 11. Still another embodiment of the inventionis performing the co-culturing of the hematopoietic progenitor cells andthe lymphoreticular stromal cells in an environment that is freealtogether of stromal cell conditioned medium and exogenously addedhematopoietic growth factors that promote hematopoietic cellmaintenance, expansion and/or differentiation, other than serum.

As will be understood, according to the invention, it is possible now toco-culture hematopoietic progenitor cells and lymphoreticular stromalcells (that may or may not include antigen presenting cells), in anenvironment that is free of exogenously added hematopoietic growthfactors that promote hematopoietic cell maintenance, expansion and/ordifferentiation for as little as 7 days and to obtain large numbers ofdifferentiated progeny of a specific lineage.

According to any of the foregoing embodiments, the method of theinvention can include co-culturing of the hematopoietic progenitorcells, the lymphoreticular stromal cells, and/or the antigen presentingcells with an exogenously added agent selected from the group consistingof stromal cell conditioned medium, and a hematopoietic growth factorthat promotes hematopoietic cell maintenance, expansion and/ordifferentiation, and influences cell localization. In certainembodiments, the hematopoietic growth factor that promotes hematopoieticcell maintenance, expansion and/or differentiation, and influences celllocalization, may be an agent that includes interleukin 3, interleukin6, interleukin 7, interleukin 11, interleukin 12, stem cell factor,FLK-2 ligand, FLT-2 ligand, Epo, Tpo, GMCSF, GCSF, Oncostatin M, andMCSF.

According to another aspect of the invention, a method for in vivomaintenance, expansion and/or differentiation of hematopoieticprogenitor cells is provided. The method involves implanting into asubject a porous, solid matrix having seeded therein hematopoieticprogenitor cells (which may include their progeny) and lymphoreticularstromal cells. The porous matrix has interconnected pores of a pore sizesufficient to permit the cells to grow throughout the matrix and is anopen cell porous matrix having a percent open space of at least 50%, andpreferably at least 75%. Various embodiments are provided, wherein theporous solid matrix has one or more of the preferred characteristics asdescribed above.

In certain embodiments, hematopoietic progenitor cells (that may includeprogeny) and lymphoreticular stromal cells are attached to the matrix byintroducing in vitro an amount of hematopoietic progenitor cells and anamount of lymphoreticular stromal cells into the porous solid matrix,and co-culturing the hematopoietic progenitor cells in an environmentthat is free of stromal cell conditioned medium and free of exogenouslyadded hematopoietic growth factors that promote hematopoietic cellmaintenance, expansion and/or differentiation, other than serum. Variousother embodiments are provided, wherein the co-culturing is performedunder conditions as described above. In yet other embodiments, theporous solid matrix having seeded hematopoietic progenitor cells (thatmay include progeny) and lymphoreticular stromal cells is impregnatedwith a gelatinous agent that occupies pores of the matrix.

According to one aspect of the invention, a method for inducing T celltolerance, is provided. The method involves producing lymphoidtissue-specific cells according to any of the foregoing co-culturemethods of the invention that involve the co-culture of cells thatinclude non-autologous cells, under conditions sufficient to induce theformation of T cells and/or T cell progenitors and to inhibit immuneactivation of the formed cells.

According to yet another aspect of the invention, a method for treatinga subject to enhance immune tolerance in the subject, is provided. Themethod involves administering to a subject in need of such treatment anamount of lymphoid tissue-specific cells produced according to any ofthe foregoing co-culture methods of the invention that involve theco-culture of cells that may include non-autologous cells, wherein theamount of lymphoid tissue-specific cells is sufficient to enhance in thesubject immune tolerance to an autologous or a non-autologous antigen.Various embodiments are provided wherein preferred cell types and porousmatrix are as described elsewhere herein (see, e.g., below).

According to still another aspect of the invention, a method forinducing T cell reactivity, is provided. The method involves producinglymphoid tissue-specific cells according to any of the foregoingco-culture methods of the invention that involve the co-culture of cellsthat may include autologous and/or non-autologous cells, in the presenceof at least one antigen, under conditions sufficient to induce formationof T cells or T cell progenitors having specificity for the at least oneantigen. In important embodiments, the at least one antigen is added tothe co-culture in a further step after formation of T cells or T cellprogenitors.

In certain embodiments, the hematopoietic progenitor cells may bepluripotent stem cells, multipotent progenitor cells and/or progenitorcells committed to specific hematopoietic lineages.

The hematopoietic progenitor cells may be derived from a tissue such asbone marrow, peripheral blood (including mobilized peripheral blood),umbilical cord blood, placental blood, fetal liver, embryonic cells(including embryonic stem cells), aortal-gonadal-mesonephros derivedcells, and lymphoid soft tissue. Lymphoid soft tissue includes thethymus, spleen, liver, lymph node, skin, tonsil and/or Peyer's patches.In other embodiments, the lymphoreticular stromal cells may be alsoderived from at least one of the foregoing lymphoid soft tissues. Inpreferred embodiments, the lymphoreticular stromal cells are thymicstromal cells and the multipotent progenitor cells and/or committedprogenitor cells are committed to a T cell lineage. In otherembodiments, the hematopoietic progenitor cells and/or thelymphoreticular stromal cells may be genetically altered.

In important embodiments, antigen presenting cells may be added to theco-culture. Antigen presenting cells include cells such as dendriticcells, monocytes/macrophages, Langerhans cells, Kupfer cells, microglia,alveolar macrophages and B cells. In other embodiments, the antigenpresenting cells are derived from hematopoietic progenitor cells invitro. Various embodiments are provided, wherein the hematopoieticprogenitor cells, the lymphoreticular stromal cells, and the poroussolid matrix have one or more of the preferred characteristics asdescribed above, and the cells are cultured as described above. Theantigen presenting cells may be derived from hematopoietic progenitorcells in vitro. In important embodiments the antigen presenting cellsare mature. In further embodiments, the method further comprisesadministering a co-stimulatory agent to the co-culture. Preferredco-stimulatory agents include lymphocyte function associated antigen 3(LFA-3), CD2, CD40, CD80/B7-1, CD86/B7-2, OX-2, CD70, and CD82.

In yet another aspect of the invention, a solid porous matrix isprovided wherein hematopoietic progenitor cells, with or without theirprogeny, and lymphoreticular stromal cells are attached to the solidporous matrix. The lymphoreticular stromal cells are present in anamount sufficient to support the growth and differentiation ofhematopoietic progenitor cells. In certain embodiments, thehematopoietic progenitor cells are attached to the lymphoreticularstromal cells. In further embodiments, the solid porous matrix mayinclude antigen presenting cells (progeny and/or nonprogeny). Preferablythe antigen presenting cells are mature. In yet further embodiments, theporous matrix further comprises at least one antigen. The porous matrixcan be one that is an open cell porous matrix having a percent openspace of at least 50%, and preferably at least 75%. In one embodimentthe porous solid matrix has pores defined by interconnecting ligamentshaving a diameter at midpoint, on average, of less than 150 μm.Preferably the porous solid matrix is a metal-coated reticulated opencell foam of carbon containing material, the metal coating beingselected from the group consisting of tantalum, titanium, platinum(including other metals of the platinum group), niobium, hafnium,tungsten, and combinations thereof. In preferred embodiments, whetherthe porous solid matrix is metal-coated or not, the matrix is coatedwith a biological agent selected from the group consisting of collagens,fibronectins, laminins, integrins, angiogenic factors, anti-inflammatoryfactors, glycosaminoglycans, vitrogen, antibodies and fragments thereof,functional equivalents of these factors, and combinations thereof. Mostpreferably the metal coating is tantalum coated with a biological agent.In certain other embodiments the porous solid matrix having seededhematopoietic progenitor cells and lymphoreticular stromal cells, isimpregnated with a gelatinous agent that occupies pores of the matrix.

In a further aspect of the invention, a method for identifying an agentsuspected of affecting hematopoietic cell development, is provided. Themethod involves introducing an amount of hematopoietic progenitor cellsand an amount of lymphoreticular stromal cells into a porous, solidmatrix having interconnected pores of a pore size sufficient to permitthe hematopoietic progenitor cells and the lymphoreticular stromal cellsto grow throughout the matrix, co-culturing the hematopoietic progenitorcells and the lymphoreticular stromal cells in the presence of at leastone candidate agent suspected of affecting hematopoietic celldevelopment (in a test co-culture), and determining whether the at leastone candidate agent affects hematopoietic cell development in the testco-culture by comparing the test co-culture hematopoietic celldevelopment to a control co-culture, whereby hematopoietic progenitorcells and lymphoreticular stromal cells are co-cultured in the absenceof the at least one candidate agent. Various embodiments are provided,wherein the hematopoietic progenitor cells, the lymphoreticular stromalcells, and the porous solid matrix have one or more of the preferredcharacteristics as described above, and the cells are cultured asdescribed above. In certain embodiments, hematopoietic progenitor celldevelopment includes hematopoietic progenitor cell maintenance,expansion, differentiation toward a specific cell lineage, and/orcell-death (including apoptosis). In preferred embodiments thelymphoreticular stromal cells are thymic stromal cells.

In another aspect of the invention, a method for isolating from a cellculture an agent suspected of affecting hematopoietic cell development,is provided. The method involves introducing an amount of hematopoieticprogenitor cells and an amount of lymphoreticular stromal cells into aporous, solid matrix having interconnected pores of a pore sizesufficient to permit the hematopoietic progenitor cells and thelymphoreticular stromal cells to grow throughout the matrix,co-culturing the hematopoietic progenitor cells and the lymphoreticularstromal cells, obtaining a test-supernatant from the co-culture,comparing the test-supernatant to a control-supernatant, and obtaining asubfraction of the test-supernatant that contains an agent suspected ofaffecting hematopoietic cell development that is absent from thecontrol-supernatant. In certain embodiments the agent suspected ofaffecting hematopoietic cell development may be present in thecontrol-supernatant and absent from the test-supernatant. In otherembodiments, the agent suspected of affecting hematopoietic celldevelopment in one supernatant may be different to an agent suspected ofaffecting hematopoietic cell development in the other supernatant (e.g.,in size, via a post-translational modification, in an alternativelyspliced variant form, etc.). Various embodiments are provided, whereinthe hematopoietic progenitor cells, the lymphoreticular stromal cells,and the porous solid matrix have one or more of the preferredcharacteristics as described above, and the cells are cultured asdescribed above. In certain embodiments, hematopoietic progenitor celldevelopment includes hematopoietic progenitor cell maintenance,expansion, differentiation toward a specific cell lineage, and/orcell-death (including apoptosis). In preferred embodiments, thelymphoreticular stromal cells are thymic stromal cells. In certain otherembodiments, the control culture system of the prior art (where thecontrol-supernatant can be obtained from) is the one described in U.S.Pat. No. 5,677,139 by Johnson et al.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the differentiation of human CD34⁺ progenitor cells into Tcells, in co-culture with murine thymic stroma cells on athree-dimensional matrix; the data in FIG. 1(a) shows the acquisition ofCD2 and the down-regulation of the hematopoietic progenitor cell markerCD34; the data in FIG. 1(b) shows the discrete populations of SP CD4⁺and SP CD8⁺ cells, including their DP CD4^(+CD)8⁺ precursors; the datain FIGS. 1(c and d) shows that all CD4⁺(c) and CD8⁺(d) cellsco-expressed CD3.

FIG. 2 shows the intrasample variability in numbers of T cells generatedin a co-culture system of the invention.

FIG. 3 shows the intersample variability in numbers of T cells generatedin a co-culture system of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the unexpected discovery that hematopoieticprogenitor cells co-cultured with lymphoreticular stromal cells in aporous solid scaffold, without the addition of exogenous growth agents,generate at a fast rate an unexpectedly high number of functional,differentiated progeny of a lymphoid tissue-specific lineage. Alsosurprising, according to the invention, has been the discovery thatlesser amounts of nonlymphoid cells (i.e. myelo-monocytic cells) aregenerated from the co-culture of hematopoietic progenitor cells andlymphoreticular stromal cells in a porous solid scaffold of theinvention when compared to existing technology. Thus, the presentinvention, and in contrast to what has been previously achieved in theart, permits for the rapid generation of a large number ofdifferentiated, lymphoid-specific cells from a relatively small numberof hematopoietic progenitor cells.

Methods of the invention are therefore useful inter alia forestablishing immunocompetence in patients suffering from animmunodeficiency, e.g., a T cell or B cell deficiency, e.g., a thymicbased immunodeficiency, e.g., a congenital immunodeficiency due tothymic aplasia or dysfunction, an acquired immune disorder, e.g., AIDS,immunoincompetence resulting form a neoplastic disease, orimmunoincompetence resulting from a medical procedure, e.g.,chemotherapy, immunocompetence in response to an antigen, etc. Methodsof generating immune cells in vitro and/or or ex vivo that could be usedin transplantation, implantation, autoimmune diseases, and/or infectiousdiseases are also contemplated.

The invention in one aspect involves culturing hematopoietic cells in aporous solid matrix, in the absence of exogenous growth agents, toproduce lymphoid tissue origin (lymphoid tissue-specific) cells.

A porous, solid matrix, is defined as a three-dimensional structure with“sponge-like” continuous pores forming an interconnecting network. Thematrix can be rigid or elastic, and it provides a scaffold upon whichcells can grow throughout. Its pores are interconnected and provide thecontinuous network of channels extending through the matrix and alsopermit the flow of nutrients throughout. A preferred matrix is an opencell foam matrix having a percent open space of at least 50% andpreferably 75%. Thus, it is preferred that the open space comprise themajority of the matrix. This is believed to maximize cell migration,cell-cell contact, space for cell growth and accessibility to nutrients.It is preferred that the porous matrix be formed of a reticulated matrixof ligaments which at their center point are less than 150 μm indiameter, preferably 60 μm, whereby a cell can reside on or interactwith a portion of the ligament. Preferably, the average pore diameter ison the order of 300 μm, which resembles cancellous bone. Suitablematrices can be obtained using a number of different methods. Examplesof such methods include solvent casting or extraction of polymers, tracketching of a variety of materials, foaming of a polymer, thereplamineform process for hydroxyapatite, and other methodologies wellknown to those of ordinary skill in the art. The materials employed canbe natural or synthetic, including biological materials such asproteins, hyaluronic acids, synthetic polymers such as polyvinylpyrolidones, polymethylmethacrylate, methyl cellulose, polystyrene,polypropylene, polyurethane, ceramics such as tricalcium phosphate,calcium aluminate, calcium hydroxyapatite and ceramic-reinforced orcoated polymers. If the starting material for the scaffold is not metal,a metal coating can be applied to the three-dimensional matrix. Metalcoatings provide further structural support and/or cell growth andadhesive properties to the matrix. Preferred metals used as coatingscomprise tantalum, titanium, platinum and metals in the same elementgroup as platinum, niobium, hafnium, tungsten, and combinations ofalloys thereof. Coating methods for metals include a process such as CVD(Chemical Vapor Deposition).

The preferred matrix, refered to herein throughout as Cellfoam(Cytomatrix, Woburn, Mass.), is described in detail in U.S. Pat. No.5,282,861, and is incorporated herein by reference. More specifically,the preferred matrix is a reticulated open cell substrate formed by alightweight, substantially rigid foam of carbon-containing materialhaving open spaces defined by an interconnecting network, wherein saidfoam material has interconnected continuous channels, and a thin film ofmetallic material deposited onto the reticulated open cell substrate andcovering substantially all of the interconnecting network to form acomposite porous biocompatible material creating a porous microstructuresimilar to that of natural cancellous bone.

Additionally, such matrices can be coated with biological agents whichcan promote cell adhesion for the cultured hematopoietic progenitorcells, allowing for improved migration, growth and proliferation.Moreover, when these matrices are used for the in vivo maintenance,expansion and/or differentiation of hematopoietic progenitor cells(i.e., when the matrices with the cells are implanted into a subject,-see also discussion below), biological agents that promote angiogenesis(vascularization) and biological agents that prevent/reduce inflammationmay also be used for coating of the matrices. Preferred biologicalagents comprise collagens, fibronectins, laminins, integrins, angiogenicfactors, anti-inflammatory factors, glycosaminoglycans, vitrogen,antibodies and fragments thereof, functional equivalents of theseagents, and combinations thereof.

Angiogenic factors include platelet derived growth factor (PDGF),vascular endothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), bFGF-2, leptins, plasminogen activators (tPA, uPA),angiopoietins, lipoprotein A, transforming growth factor-β, bradykinin,angiogenic oligosaccharides (e.g., hyaluronan, heparan sulphate),thrombospondin, hepatocyte growth factor (also known as scatter factor)and members of the CXC chemokine receptor family. Anti-inflammatoryfactors comprise steroidal and non-steroidal compounds and examplesinclude: Alclofenac; Alclometasone Dipropionate; Algestone Acetonide;Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit ; Salcolex ; Salnacedin; Salsalate ;Sanguinarium Chloride; Seclazone ; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine ; Tiopinac ;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

In certain embodiments of the invention the porous solid matrix havingseeded hematopoietic progenitor cells, with or without their progeny,and lymphoreticular stromal cells is impregnated with a gelatinous agentthat occupies pores of the matrix. The hematopoietic progenitor cells,with or without their progeny, and/or the lymphoreticular stromal cellscan be seeded prior to, substantially at the same time as, or followingimpregnation (or infiltration) with a gelatinous agent. For example, thecells may be mixed with the agent and seeded at the same time as the theimpregnation of the matrix with the agent. In some embodiments, thecells are seeded onto the porous solid matrix prior to application ofthe agent. In certain embodiments the lymphoreticular stromal cells areseeded in a similar manner. A person of ordinary skill in the art caneasily determine seeding conditions. Preferably the lymphoreticularstromal cells are seeded prior to the hematopoietic progenitor cells andprior to impregnation with the agent.

“Impregnation” with a gelatinous agent can serve, inter alia, to containthe cells within the matrix, or to help maintain and/or enhance cellattachment onto the matrix. The “gelatinous” agent may be one that canbe maintained in a fluid state initially (i.e. gelable), and after itsapplication into the matrix, be gelatinized in situ in the matrix. Suchgelatinization may occur in a number of different ways, includingaltering the agent's temperature, irradiating the agent with an energysource (e.g., light), etc. The “gelatinous” agent also is characterizedby its ability to allow the nutrients of the growth media to reach thecells throughout the matrix. Exemplary “gelatinous” agents includecellulosic polysaccharides (such as cellulose, hemicellulose,methylcellulose, and the like), agar, agarose, albumin, algal mucin,mucin, mucilage, collagens, glycosaminoglycans, and proteoglycans(including their sulphated forms). In certain embodiments, thegelatinous agent may impregnate the matrix completely, in someembodiments partially, and in other embodiments minimally, serving onlyas a coating of all or some of the outer surfaces of the matrix. Inimportant embodiments where gelatinous agents are employed, the“gelatinous” agent is methylcellulose and the impregnation is complete.

According to the invention, hematopoietic progenitor cells andlymphoreticular stromal cells are co-cultured in one of the foregoingporous solid matrices, in the absence of exogenous growth agents, toproduce lymphoid tissue origin (lymphoid tissue-specific) cells.“Lymphoid tissue origin” (lymphoid tissue-specific) cells, as usedherein, refer to cells that may be produced in vitro or in vivoaccording to the invention, and are substantially similar (e.g., inproperties and function) to the cells produced naturally in vivo fromorgans and tissues that include the bone marrow, thymus, lymph nodes,spleen and mucosal associated lymphoid tissue (unencapsulated tissuelining the respiratory, alimentary and genito-urinary tracts).

“Hematopoietic progenitor cells” as used herein refers to immature bloodcells having the capacity to self-renew and to differentiate into themore mature blood cells (also described herein as “progeny”) comprisinggranulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets), andmonocytes (e.g., monocytes, macrophages). It is known in the art thatsuch cells may or may not include CD34⁺ cells. CD34⁺ cells are immaturecells present in the “blood products” described below, express the CD34cell surface marker, and are believed to include a subpopulation ofcells with the “progenitor cell” properties defined above. It is wellknown in the art that hematopoietic progenitor cells include pluripotentstem cells, multipotent progenitor cells (e.g., a lymphoid stem cell),and/or progenitor cells committed to specific hematopoietic lineages.The progenitor cells committed to specific hematopoietic lineages may beof T cell lineage, B cell lineage, dendritic cell lineage, Langerhanscell lineage and/or lymphoid tissue-specific macrophage cell lineage.

The hematopoietic progenitor cells can be obtained from blood products.A “blood product” as used in the present invention defines a productobtained from the body or an organ of the body containing cells ofhematopoietic origin. Such sources include unfractionated bone marrow,umbilical cord, peripheral blood, liver, thymus, lymph and spleen. Itwill be apparent to those of ordinary skill in the art that all of theaforementioned crude or unfractionated blood products can be enrichedfor cells having “hematopoietic progenitor cell” characteristics in anumber of ways. For example, the blood product can be depleted from themore differentiated progeny. The more mature, differentiated cells canbe selected against, via cell surface molecules they express.Additionally, the blood product can be fractionated selecting for CD34⁺cells. As mentioned earlier, CD34 +cells are thought in the art toinclude a subpopulation of cells capable of self-renewal andpluripotentiality. Such selection can be accomplished using, forexample, commercially available magnetic anti-CD34 beads (Dynal, LakeSuccess, N.Y.). Unfractionated blood products can be obtained directlyfrom a donor or retrieved from cryopreservative storage.

The cells co-cultured with the hematopoietic progenitor cells accordingto the methods of the invention are lymphoreticular stromal cells.“Lymphoreticular stromal cells” as used herein may include, but are notlimited to, all cell types present in a lymphoid tissue which are notlymphocytes or lymphocyte precursors or progenitors, e.g., epithelialcells, endothelial cells, mesothelial cells, dendritic cells,splenocytes and macrophages. Lymphoreticular stromal cells also includecells that would not ordinarily function as lymphoreticular stromalcells, such as fibroblasts, which have been genetically altered tosecrete or express on their cell surface the factors necessary for themaintenance, growth and/or differentiation of hematopoietic progenitorcells, including their progeny. Lymphoreticular stromal cells arederived from the disaggregation of a piece of lymphoid tissue (seediscussion below and the Examples). Such cells according to theinvention are capable of supporting in vitro the maintenance, growthand/or differentiation of hematopoietic progenitor cells, includingtheir progeny. By “lymphoid tissue” it is meant to include bone marrow,peripheral blood (including mobilized peripheral blood), umbilical cordblood, placental blood, fetal liver, embryonic cells (includingembryonic stem cells), aortal-gonadal-mesonephros derived cells, andlymphoid soft tissue. “Lymphoid soft tissue” as used herein includes,but is not limited to, tissues such as thymus, spleen, liver, lymphnode, skin, tonsil, adenoids and Peyer's patch, and combinationsthereof.

Lymphoreticular stromal cells provide the supporting microenvironment inthe intact lymphoid tissue for the maintenance, growth and/ordifferentiation of hematopoietic progenitor cells, including theirprogeny. The microenvironment includes soluble and cell surface factorsexpressed by the various cell types which comprise the lymphoreticularstroma. Generally, the support which the lymphoreticular stromal cellsprovide may be characterized as both contact-dependent andnon-contact-dependent.

Lymphoreticular stromal cells may be autologous (“self”) ornon-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic)with respect to hematopoietic progenitor cells or antigen presentingcells. “Autologous,” as used herein, refers to cells from the samesubject. “Allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison. “Syngeneic,”as used herein, refers to cells of a different subject that aregenetically identical to the cell in comparison. “Xenogeneic,” as usedherein, refers to cells of a different species to the cell incomparison. Lymphoreticular stroma cells may be obtained from thelymphoid tissue of a human or a non-human subject at any time after theorgan/tissue has developed to a stage (i.e., the maturation stage) atwhich it can support the maintenance growth and/or differentiation ofhematopoietic progenitor cells. The stage will vary betweenorgans/tissues and between subjects. In primates, for example, thematuration stage of thymic development is achieved during the secondtrimester. At this stage of development the thymus can produce peptidehormones such as thymulin, α₁ and β₄-thymosin, and thymopoietin, as wellas other factors required to provide the proper microenvironment for Tcell differentiation. The different maturation stages for the differentorgans/tissues and between different subjects are well known in the art.

The lymphoid tissue from which lymphoreticular stromal cells are derivedusually determines the lineage-commitment hematopoietic progenitor cellsundertake, resulting in the lineage-specificity of the differentiatedprogeny. In certain embodiments, the lymphoreticular stromal cells arethymic stromal cells and the multipotent progenitor cells and/orcommitted progenitor cells are committed to a T cell lineage. In otherembodiments, the lymphoreticular stromal cells may be splenic stromalcells and the multipotent progenitor cells and/or committed progenitorcells are committed to a B cell lineage. Also surprising, according tothe invention, has been the discovery that the highest yield ofdifferentiated progeny occurs when human hematopoietic progenitor cellsare cultured in the presence of xenogeneic (non-human) lymphoreticularstromal cells. Preferably the xenogeneic lymphoreticular stromal cellsare of murine origin.

Unexpectedly, it has also been discovered that lesser amounts ofnonlymphoid-specific cells (i.e. myelo-monocytic cells) are generatedfrom the foregoing co-cultures when compared to existing methodology. Inother words, more homogeneous differentiation of cells with fewercontaminant cell types (nonlymphoid) is observed form cultures of thepresent invention on Cellfoam, enabling the preservation of immatureprogenitors (CD34⁺ cells) while promoting the differentiation of moremature T progeny.

Various other embodiments are provided, wherein the lymphoreticularstromal cells may be genetically altered. The lymphoreticular stromalcells may be transfected with exogenous DNA that encodes, for example,one of the hematopoietic growth factors described above (see fibroblastdiscussion above).

As mentioned earlier, lymphoreticular stromal cells are derived from thedisaggregation of a piece of lymphoid tissue, forming cell suspensions.Preferably, single cell suspensions are generated. These lymphoreticularstromal cell suspensions may be used directly, or made non-mitotic byprocedures standard in the tissue culture art. Examples of such methodsare irradiation of lymphoreticular stromal cells with a gamma-ray sourceor incubation of the cells with mitomycin C for a sufficient amount oftime to render the cells mitotically inactive. Mitotic inactivation ispreferred when the lymphoreticular stromal cells are of human origin (toeliminate progenitor cells that may be present in the suspension). Thelymphoreticular stromal cells may then be seeded into athree-dimensional matrix of the invention and permitted to attach to asurface of the porous, solid matrix. It should be noted that thelymphoreticular stromal cells may alternatively be cryopreserved forlater use or for storage and shipment to remote locations, such as foruse in connection with the sale of kits. Cryopreservation of cellscultured in vitro is well established in the art. Subsequent toisolation (and/or mitotic inactivation) of a cell sample, cells may becryopreserved by first suspending the cells in a cryopreservation mediumand then gradually freezing the cell suspension. Frozen cells aretypically stored in liquid nitrogen or at an equivalent temperature in amedium containing serum and a cryopreservative such as dimethylsulfoxide.

The co-culture of the hematopoietic progenitor cells (and progenythereof) with lymphoreticular stromal cells, preferably occurs underconditions sufficient to produce a percent increase in the number oflymphoid tissue origin cells deriving from the hematopoietic progenitorcells. The conditions used refer to a combination of conditions known inthe art (e.g., temperature, CO₂ and O₂ content, nutritive media,time-length, etc.). The time sufficient to increase the number of cellsis a time that can be easily determined by a person skilled in the art,and can vary depending upon the original number of cells seeded. Theamounts of hematopoietic progenitor cells and lymphoreticular stromalcells initially introduced (and subsequently seeded) into the poroussolid matrix may vary according to the needs of the experiment. Theideal amounts can be easily determined by a person skilled in the art inaccordance with needs. Preferably, the lymphoreticular stromal cellswould form a confluent layer onto the matrix. Hematopoietic progenitorcells may be added at different numbers. As an example, discoloration ofthe media over a certain period of time can be used as an indicator ofconfluency. Additionally, and more precisely, different numbers ofhematopoietic progenitor cells or volumes of the blood product can becultured under identical conditions, and cells can be harvested andcounted over regular time intervals, thus generating the “controlcurves”. These “control curves” can be used to estimate cell numbers insubsequent occasions (see the Examples section).

The conditions for determining colony forming potential are similarlydetermined. Colony forming potential is the ability of a cell to formprogeny. Assays for this are well known to those of ordinary skill inthe art and include seeding cells into a semi-solid matrix, treatingthem with growth factors, and counting the number of colonies.

In preferred embodiments of the invention, the hematopoietic progenitorcells may be harvested. “Harvesting” hematopoietic progenitor cells isdefined as the dislodging or separation of cells from the matrix. Thiscan be accomplished using a number of methods, such as enzymatic andnon-enzymatic, centrifugal, electrical or by size, or the one preferredin the present invention, by flushing of the cells using the media inwhich the cells are incubated. The cells can be further collected,separated, and further expanded generating even larger populations ofdifferentiated progeny.

As mentioned above, the hematopoietic progenitor cells, and progenythereof, can be genetically altered. Genetic alteration of ahematopoietic progenitor cell includes all transient and stable changesof the cellular genetic material which are created by the addition ofexogenous genetic material. Examples of genetic alterations include anygene therapy procedure, such as introduction of a functional gene toreplace a mutated or nonexpressed gene, introduction of a vector thatencodes a dominant negative gene product, introduction of a vectorengineered to express a ribozyme and introduction of a gene that encodesa therapeutic gene product. Natural genetic changes such as thespontaneous rearrangement of a T cell receptor gene without theintroduction of any agents are not included in this concept. Exogenousgenetic material includes nucleic acids or oligonucleotides, eithernatural or synthetic, that are introduced into the hematopoieticprogenitor cells. The exogenous genetic material may be a copy of thatwhich is naturally present in the cells, or it may not be naturallyfound in the cells. It typically is at least a portion of a naturallyoccurring gene which has been placed under operable control of apromoter in a vector construct.

The invention involves the unexpected discovery that hematopoieticprogenitor cells can be more efficiently genetically altered if thegenetic alteration occurs while the hematopoietic progenitor cells areon and within a solid porous matrix as described above.

Various techniques may be employed for introducing nucleic acids intocells. Such techniques include transfection of nucleic acid-CaPO₄precipitates, transfection of nucleic acids associated with DEAE,transfection with a retrovirus including the nucleic acid of interest,liposome mediated transfection, and the like. For certain uses, it ispreferred to target the nucleic acid to particular cells. In suchinstances, a vehicle used for delivering a nucleic acid according to theinvention into a cell (e.g., a retrovirus, or other virus; a liposome)can have a targeting molecule attached thereto. For example, a moleculesuch as an antibody specific for a surface membrane protein on thetarget cell or a ligand for a receptor on the target cell can be boundto or incorporated within the nucleic acid delivery vehicle. Forexample, where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

In the present invention, the preferred method of introducing exogenousgenetic material into hematopoietic cells is by transducing the cells insitu on the matrix using replication-deficient retroviruses.Replication-deficient retroviruses are capable of directing synthesis ofall virion proteins, but are incapable of making infectious particles.Accordingly, these genetically altered retroviral vectors have generalutility for high-efficiency transduction of genes in cultured cells, andspecific utility for use in the method of the present invention.Retroviruses have been used extensively for transferring geneticmaterial into cells. Standard protocols for producingreplication-deficient retroviruses (including the steps of incorporationof exogenous genetic material into a plasmid, transfection of apackaging cell line with plasmid, production of recombinant retrovirusesby the packaging cell line, collection of viral particles from tissueculture media, and infection of the target cells with the viralparticles) are provided in the art.

The major advantage of using retroviruses is that the viruses insertefficiently a single copy of the gene encoding the therapeutic agentinto the host cell genome, thereby permitting the exogenous geneticmaterial to be passed on to the progeny of the cell when it divides. Inaddition, gene promoter sequences in the LTR region have been reportedto enhance expression of an inserted coding sequence in a variety ofcell types. The major disadvantages of using a retrovirus expressionvector are (1) insertional mutagenesis, i.e., the insertion of thetherapeutic gene into an undesirable position in the target cell genomewhich, for example, leads to unregulated cell growth and (2) the needfor target cell proliferation in order for the therapeutic gene carriedby the vector to be integrated into the target genome. Despite theseapparent limitations, delivery of a therapeutically effective amount ofa therapeutic agent via a retrovirus can be efficacious if theefficiency of transduction is high and/or the number of target cellsavailable for transduction is high.

Yet another viral candidate useful as an expression vector fortransformation of hematopoietic cells is the adenovirus, adouble-stranded DNA virus. Like the retrovirus, the adenovirus genome isadaptable for use as an expression vector for gene transduction, i.e.,by removing the genetic information that controls production of thevirus itself. Because the adenovirus functions usually in anextrachromosomal fashion, the recombinant adenovirus does not have thetheoretical problem of insertional mutagenesis. On the other hand,adenoviral transformation of a target hematopoietic cell may not resultin stable transduction. However, more recently it has been reported thatcertain adenoviral sequences confer intrachromosomal integrationspecificity to carrier sequences, and thus result in a stabletransduction of the exogenous genetic material.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable vectors are available for transferring exogenous geneticmaterial into hematopoietic cells. The selection of an appropriatevector to deliver a therapeutic agent for a particular conditionamenable to gene replacement therapy and the optimization of theconditions for insertion of the selected expression vector into thecell, are within the scope of one of ordinary skill in the art withoutthe need for undue experimentation. The promoter characteristically hasa specific nucleotide sequence necessary to initiate transcription.Optionally, the exogenous genetic material further includes additionalsequences (i.e., enhancers) required to obtain the desired genetranscription activity. For the purpose of this discussion an “enhancer”is simply any nontranslated DNA sequence which works contiguous with thecoding sequence (in cis) to change the basal transcription leveldictated by the promoter. Preferably, the exogenous genetic material isintroduced into the hematopoietic cell genome immediately downstreamfrom the promoter so that the promoter and coding sequence areoperatively linked so as to permit transcription of the coding sequence.A preferred retroviral expression vector includes an exogenous promoterelement to control transcription of the inserted exogenous gene. Suchexogenous promoters include both constitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a gene under the control of aconstitutive promoter is expressed under all conditions of cell growth.Exemplary constitutive promoters include the promoters for the followinggenes which encode certain constitutive or “housekeeping” functions:hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase(DHFR) (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630(1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvatekinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc.Natl. Acad. Sci. USA 86: 10006-10010 (1989)), and other constitutivepromoters known to those of skill in the art. In addition, many viralpromoters function constitutively in eukaryotic cells. These include:the early and late promoters of SV40; the long terminal repeats (LTRS)of Moloney Leukemia Virus and other retroviruses; and the thymidinekinase promoter of Herpes Simplex Virus, among many others. Accordingly,any of the above-referenced constitutive promoters can be used tocontrol transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressedonly or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). Induciblepromoters include responsive elements (REs) which stimulatetranscription when their inducing factors are bound. For example, thereare REs for serum factors, steroid hormones, retinoic acid and cyclicAMP. Promoters containing a particular RE can be chosen in order toobtain an inducible response and in some cases, the RE itself may beattached to a different promoter, thereby conferring inducibility to therecombinant gene. Thus, by selecting the appropriate promoter(constitutive versus inducible; strong versus weak), it is possible tocontrol both the existence and level of expression of a therapeuticagent in the genetically modified hematopoietic cell. Selection andoptimization of these factors for delivery of a therapeuticallyeffective dose of a particular therapeutic agent is deemed to be withinthe scope of one of ordinary skill in the art without undueexperimentation, taking into account the above-disclosed factors and theclinical profile of the patient.

In addition to at least one promoter and at least one heterologousnucleic acid encoding the therapeutic agent, the expression vectorpreferably includes a selection gene, for example, a neomycin resistancegene, for facilitating selection of hematopoietic cells that have beentransfected or transduced with the expression vector. Alternatively, thehematopoietic cells are transfected with two or more expression vectors,at least one vector containing the gene(s) encoding the therapeuticagent(s), the other vector containing a selection gene. The selection ofa suitable promoter, enhancer, selection gene and/or signal sequence(described below) is deemed to be within the scope of one of ordinaryskill in the art without undue experimentation.

The selection and optimization of a particular expression vector forexpressing a specific gene product in an isolated hematopoietic cell isaccomplished by obtaining the gene, preferably with one or moreappropriate control regions (e.g., promoter, insertion sequence);preparing a vector construct comprising the vector into which isinserted the gene; transfecting or transducing cultured hematopoieticcells in vitro with the vector construct; and determining whether thegene product is present in the cultured cells.

TABLE 1 Human Gene Therapy Protocols Approved by RAC: 1990-1994 Severecombined Autologous lymphocytes transduced 7/31/90 immune deficiencywith human ADA gene (SCID) due to ADA deficiency Advanced cancerTumor-infiltrating lymphocytes trans- 7/31/90 duced with tumor necrosisfactor gene Advanced cancer Immunization with autologous cancer10/07/91  cells transduced with tumor necrosis factor gene Advancedcancer Immunization with autologous cancer 10/07/91  cells transducedwith interleukin-2 gene Asymptomatic Murine Retro viral vector encoding6/07/93 patients infected HIV-1 genes [HIV-IT(V)] with HIV-1 AIDSEffects of a transdominant form of 6/07/93 rev gene on AIDS interventionAdvanced cancer Human multiple-drug resistance 6/08/93 (MDR) genetransfer HIV infection Autologous lymphocytes transduced 9/10/93 withcatalytic ribozyme that cleaves HIV-1 RNA (Phase I study) MetastaticGenetically engineered autologous 9/10/93 melanoma tumor vaccinesproducing inter- leukin-2 HIV infection Murine Retro viral vectorencoding 12/03/93  HIV-IT(V) genes (open label Phase I/II trial) HIVinfection Adoptive transfer of syngeneic cyto- 3/03/94 (identical twins)toxic T lymphocytes (Phase I/II pilot study) Breast cancer Use ofmodified Retro virus to intro- 6/09/94 (chemoprotection ducechemotherapy resistance during therapy) sequences into normalhematopoietic cells (pilot study) Fanconi's anemia Retro viral mediatedgene transfer of 6/09/94 the Fanconi anemia complementation group C geneto hematopoietic progenitors Metastatic prostate Autologous humangranulocyte ORDA/NIH carcinoma macrophage-colony stimulating factor 8/03/94* gene transduced prostate cancer vaccine *(first protocol to beapproved under the accelerated review process; ORDA = Office ofRecombinate DNA Activities) Metastatic breast In vivo infection withbreast-targeted 9/12/94 cancer Retro viral vector expressing anti- sensec-fox or antisense c-myc RNA Metastatic breast Non-viral system(liposome-based) 9/12/94 cancer (refractory for delivering humaninterleukin-2 or recurrent) gene into autologous tumor cells (pilotstudy) Mild Hunter Retro viral-mediated transfer of the 9/13/94 syndromeiduronate-2-sulfatase gene into lymphocytes Advanced Use of recombinantadenovirus 9/13/94 mesothelioma (Phase I study)

The foregoing (Table 1), represent only examples of genes that can bedelivered according to the methods of the invention. Suitable promoters,enhancers, vectors, etc., for such genes are published in the literatureassociated with the foregoing trials. In general, useful genes replaceor supplement function, including genes encoding missing enzymes such asadenosine deaminase (ADA) which has been used in clinical trials totreat ADA deficiency and cofactors such as insulin and coagulationfactor VIII. Genes which affect regulation can also be administered,alone or in combination with a gene supplementing or replacing aspecific function. For example, a gene encoding a protein whichsuppresses expression of a particular protein-encoding gene can beadministered. The invention is particularly useful in delivering geneswhich stimulate the immune response, including genes encoding viralantigens, tumor antigens, cytokines (e.g. tumor necrosis factor) andinducers of cytokines (e.g. endotoxin).

Employing the culture conditions described in greater detail below, itis possible according to the invention to preserve hematopoieticprogenitor cells and to stimulate the expansion of hematopoieticprogenitor cell number and/or colony forming unit potential. Onceexpanded, the cells, for example, can be returned to the body tosupplement, replenish, etc. a patient's hematopoietic progenitor cellpopulation. This might be appropriate, for example, after an individualhas undergone chemotherapy. There are certain genetic conditions whereinhematopoietic progenitor cell numbers are decreased, and the methods ofthe invention may be used in these situations as well.

It also is possible to take the increased numbers of hematopoieticprogenitor cells produced according to the invention and stimulate themwith hematopoietic growth agents that promote hematopoietic cellmaintenance, expansion and/or differentiation, and also influence celllocalization, to yield the more mature blood cells, in vitro. Suchexpanded populations of blood cells may be applied in vivo as describedabove, or may be used experimentally as will be recognized by those ofordinary skill in the art. Such differentiated cells include thosedescribed above, as well as T cells, plasma cells, erythrocytes,megakaryocytes, basophils, polymorphonuclear leukocytes, monocytes,macrophages, eosinohils and platelets.

In all of the culturing methods according to the invention, except asotherwise provided, the media used is that which is conventional forculturing cells. Examples include RPMI, DMEM, Iscove's, etc. Typicallythese media are supplemented with human or animal plasma or serum. Suchplasma or serum can contain small amounts of hematopoietic growthfactors. The media used according to the present invention, however, candepart from that used conventionally in the prior art. In particular, ithas been discovered, surprisingly, that hematopoietic progenitor cellscan be cultured on the matrices described above for extended periods oftime without the need for adding any exogenous growth agents (other thanthose which may be contained in plasma or serum, hereinafter “serum”),without inoculating the environment of the culture with stromal cellsand without using stromal cell conditioned media. Prior to the presentinvention, at least one of the foregoing agents was believed necessaryin order to culture hematopoietic progenitor cells.

The growth agents of particular interest in connection with the presentinvention are hematopoietic growth factors. By hematopoietic growthfactors, it is meant factors that influence the survival, proliferationor differentiation of hematopoietic progenitor cells. Growth agents thataffect only survival and proliferation, but are not believed to promotedifferentiation, include the interleukins 3, 6 and 11, stem cell factorand FLT-3 ligand. Hematopoietic growth factors that promotedifferentiation include the colony stimulating factors such as GMCSF,GCSF, MCSF, Tpo, Epo, Oncostatin M, and interleukins other than IL-3, 6and 11. The foregoing factors are well known to those of ordinary skillin the art. Most are commercially available. They can be obtained bypurification, by recombinant methodologies or can be derived orsynthesized synthetically.

“Stromal cell conditioned medium” refers to medium in which theaforementioned lymphoreticular stromal cells have been incubated. Theincubation is performed for a period sufficient to allow the stromalcells to secrete factors into the medium. Such “stromal cell conditionedmedium” can then be used to supplement the culture of hematopoieticprogenitor cells promoting their proliferation and/or differentiation.

Thus, when cells are cultured without any of the foregoing agents, it ismeant herein that the cells are cultured without the addition of suchagent except as may be present in serum, ordinary nutritive media orwithin the blood product isolate, unfractionated or fractionated, whichcontains the hematopoietic progenitor cells.

According to another aspect of the invention a method for in vivomaintenance, expansion and/or differentiation of hematopoieticprogenitor cells is provided. The method involves implanting into asubject a porous solid matrix having seeded hematopoietic progenitorcells, hematopoietic progenitor cell progeny, and lymphoreticularstromal cells. Implantation of matrices similar to the matrices of theinvention is well known in the art (Stackpool, G J, et al, CombinedOrthopaedic Research Societies Meeting, Nov. 6-8, 1995, San Diego,Calif., Abstract Book p. 45; Turner, T M, et al., 21st Annual Meeting ofthe Society for Biomaterials, March 18-22, San Francisco, Calif.,Abstract Book p. 125). Such matrices are biocompatible (i.e., no immunereactivity-no rejection) and can be implanted and transplanted in anumber of different tissues of a subject. Such methods are useful in avariety of ways, including the study of hematopoietic progenitor cellmaintenance, expansion, differentiation and/or localization in vivo, ina number of different tissues of a subject, and/or between differentsubjects.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. Human hematopoietic progenitorcells and human subjects are particularly important embodiments. Asdescribed above, when the matrices of the invention are used for such invivo implantation studies, biological agents that promote angiogenesis(vascularization) and/or prevent/reduce inflammation may also be usedfor coating of the matrices. Preferred biological agents are asdescribed above. Also as described above, the hematopoietic progenitorcells are pre-seeded onto the porous solid matrix and cultured in vitroaccording to the invention, before implantation into a subject.According to the invention, an amount of the cells is introduced invitro into the porous solid matrix, and co-cultured with lymphoreticularstromal cells in an environment that is free stromal cell conditionedmedium, and exogenously added hematopoietic growth factors that promotehematopoietic cell maintenance, expansion and/or differentiation, otherthan serum. Implantation is then carried out. In certain embodiments,stromal cell conditioned medium and exogenous hematopoietic growthfactors may be added during the in vitro culture before implantation.

According to one aspect of the invention, a method for inducing T cellreactivity/activation, in vitro, is provided. Induction of T cellreactivity/activation involves co-culturing the hematopoietic progenitorcells and the lymphoreticular stromal cells in the presence of anantigen, in one of the foregoing matrices, under conditions sufficientto induce the formation of T cells and/or T cell progenitors from thehematopoietic progenitor cells having specificity for the antigen. Theforegoing conditions could easily be established by a person of ordinaryskill in the art, without undue experimentation (see also Sprent J, etal., J Immunother, 1998, 21(3):181-187; Berridge M J, Crit Rev Immunol,1997, 17(2):155-178; Owen M J, et al., Curr Opin Immunol, 1996,8(2):191-198; Whitfield J F, et al., Mol Cell Biochem, 1979,27(3):155-179; Fauci A S, et al., Ann Intern Med, 1983, 99(1):61-75).

In important embodiments, antigen presenting cells (preferably mature)are also included in the co-culture of the hematopoietic progenitorcells and the lymphoreticular stromal cells. Antigen stimulation of Tcells in the presence of APCs, induces an antigen specific response thatcan be measured using a proliferation assay or just by measuring IL-2production (see discussion below). These cells can be detected byculturing T cells with antigen at an appropriate concentration (e.g.,0.1-1.0 μM tetanus toxoid) in the presence of APCs. If antigen specificT cells are present they can be detected using the assays describedbelow under self-tolerance/anergy. Stimulation of T cells in thepresence of APCs may include co-stimulation with a co-stimulatory agent.Co-stimulatory agents include lymphocyte function associated antigen-3(LFA-3), CD2, CD40, CD80/B7-1, CD86/B7-2, OX-2, CD70, and CD82.Co-stimulatory agents may also be used in lieu of APCs, provided thatMHC class II molecules and anti-CD3 antibodies are co-administered withthe co-stimulatory agent(s).

An antigen, as used herein, falls into four classes: 1) antigens thatare characteristic of a pathogen; 2) antigens that are characteristic ofan autoimmune disease; 3) antigens that are characteristic of anallergen; and 4) antigens that are characteristic of a tumor. Antigensin general include polysaccharides, glycolipids, glycoproteins,peptides, proteins, carbohydrates and lipids from cell surfaces,cytoplasm, nuclei, mitochondria and the like.

Antigens that are characteristic of pathogens include antigens derivedfrom viruses, bacteria, parasites or fungi. Examples of importantpathogens include vibrio choleras, enterotoxigenic Escherichia coli,rotavirus, Clostridium difficile, Shigella species, Salmonella typhi,parainfluenza virus, influenza virus, Streptococcus pneumonias, Borellaburgdorferi, HIV, Streptococcus mutans, Plasmodium falciparum,Staphylococcus aureus, rabies virus and Epstein-Barr virus.

Viruses in general include but are not limited to those in the followingfamilies: picornaviridae; caliciviridae; togaviridae; flaviviridae;coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae;orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae;hepadnaviridae; parvoviridae; papovaviridae; adenoviridae;herpesviridae; and poxyviridae; and viruses including, but not limitedto, cytomegalovirus; Hepatitis A,B,C, D, E; Herpes simplex virus types 1& 2; Influenzae virus; Mumps virus; Parainfluenza 1, 2 and 3; EpsteinBarr virus; Respiratory syncytial virus; Rubella virus; Rubeola virus;Varicella-zoster virus; Vibrio Cholerae; Human immunodeficiency viruses(HIVs) and HIV peptides, including HIV-1 gag, HIV-1 env, HIV-2 gag,HIV-2 env, Nef, RT, Rev, gp120, gp41, p15, p17, p24, p7-p6, Pol, Tat,Vpr, Vif, Vpu; Hantavirus; Ebola virus; Lymphocytic ChorioMeningitisvirus; Dengue virus; Rotavirus; Human T-lymphotropic (HTLV-I); HTLV-II;Human herpesvirus-6 (HHV-6); HHV-8; Guanarito virus; Bartonellahenselae; Sin nombre virus; and Sabia virus. Exemplary cytomegalovirusepitopes include GP 33-43, NP396-404, and GP276-286. An exemplaryinfluenza epitope includes the HA peptide.

Bacteria in general include but are not limited to: P. aeruginosa;Bacillus anthracis; E. coli, Enterocytozoon bieneusi; Klebsiella sp.;Klebsiella pneumoniae; Serratia sp.; Pseudomonas sp.; P. cepacia;Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumoniae; S. aureus;Haemophilus sp.; Haemophilus Influenza; Neisseria Sp.; Neisseriagonorheae; Neisseria meningitis; Helicobacter pylori; Bacteroides sp.;Citrobacter sp.; Branhamella sp.; Salmonella sp.; Salmonella typhi;Shigella sp.; S. pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrixsp.; Lesteria sp.; Pasteurella multocida; Streptobacillus sp.; Spirillumsp.; Fusospirocheta sp.; Actinomycetes; Mycoplasma sp.; Chlamydiae sp.;Chlamydia trachomatis; Campylobacter jejuni; Cyclospora cayatanensis;Rickettsia sp.; Spirochaeta, including Treponema pallidum and Borreliasp.; Legionella sp.; Legionella pneumophila; Mycobacteria sp.;Mycobacterium tuberculosis; Ureaplasma sp.; Streptomyces sp.;Trichomonas sp.; and P. mirabilis, as well as toxins, that include, butare not limited to, Anthrax toxin (EF); Adenylate cyclase toxin; Choleraenterotoxin; E. coli LT toxin; Escherichia coli 0157:H7; Shiga toxin;Botulinum Neurotoxin Type A heavy and light chains; Botulinum NeurotoxinType B heavy and light chains; Tetanus toxin; Tetanus toxin C fragment;Diphtheria toxin; Pertussis toxin; Parvovirus B19; Staphylococcusenterotoxins; Toxic shock syndrome toxin (TSST-1); Erythrogenic toxin;and Vibrio cholerae 0139.

Parasites include but are not limited to: Ehrlichia chafeensis; Babesia;Encephalitozoon cuniculi; Encephalitozoon hellem; Schistosoms;Toxoplasma gondii; Plasmodium falciparum, P. vivax, P. ovale, P.malaria; Toxoplasma gondii; Leishmania mexicana, L. tropica, L. major,L. aethiopica, L. donovani, Trypanosoma cruzi, T. brucei, Schistosomamansoni, S. haematobium, S. japonium; Trichinella spiralis; Wuchereriabancrofti; Brugia malayli; Entamoeba histolytica; Enterobiusvermiculoarus; Taenia solium, T. saginata, Trichomonas vaginatis, T.hominis, T. tenax; Giardia lamblia; Cryptosporidum parvum; Pneumocytiscarinii, Babesia bovis, B. divergens, B. microti, Isospore belli, Lhominis; Dientamoeba fragiles; Onchocerca volvulus; Ascarislumbricoides; Necator americanis; Ancylostoma duodenale; Strongyloidesstercoralis; Capillaria philippinensis; Angiostrongylus cantonensis;Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E.multilocularis; Paragonimus westermani, P. caliensis; Chlonorchissinensis; Opisthorchis felineas, G. Viverini, Fasciola hepatica,Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; and Dermatobiahominis.

Fungi in general include but are not limited to: Cryptococcusneoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis;Histoplasfria capsulatum; Coccidioides immitis; Candida species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunninghammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

Antigens that are characteristic of autoimmune disease typically will bederived from the cell surface, cytoplasm, nucleus, mitochondria and thelike of mammalian tissues. Examples include antigens characteristic ofuveitis (e.g. S antigen), diabetes mellitus, multiple sclerosis,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, primary myxoedema, thyrotoxicosis, rheumatoid arthritis,pernicious anemia, Addison's disease, scleroderma, autoimmune atrophicgastritis, premature menopause (few cases), male infertility (fewcases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris,pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmunehaemolytic anemia, idiopathic thrombocylopenic purpura, idiopathicfeucopenia, primary biliary cirrhosis (few cases), ulcerative colitis,Siogren's syndrome, Wegener's granulomatosis, poly/dermatomyositis, anddiscold lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins,although allergens may also be low molecular weight allergenic haptensthat induce allergy after covalently combining with a protein carrier(Remington's Pharmaceutical Sciences). Allergens include antigensderived from pollens, dust, molds, spores, dander, insects and foods.Specific examples include the urushiols (pentadecylcatechol orheptadecyicatechol) of Toxicodendron species such as poison ivy, poisonoak and poison sumac, and the sesquiterpenoid lactones of ragweed andrelated plants.

Antigens that are characteristic of tumor antigens typically will bederived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and tumor mucins and glycolipids.Tumors include, but are not limited to, those from the following sitesof cancer and types of cancer: biliary tract cancer; brain cancer,including glioblastomas and medulloblastomas; breast cancer; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; hematological neoplasms, including acutelymphocytic and myelogeneous leukemia; multiple myeloma; AIDS associatesleukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms,including Bowen's disease and Paget's disease; liver cancer; lungcancer; lymphomas, including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer, including squamous cellcarcinoma; ovarian cancer, including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreas cancer;prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skincancer, including melanoma, Kaposi's sarcoma, basal cell cancer andsquamous cell cancer; testicular cancer, including germinal tumors(seminoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumorsand germ cell tumors; thyroid cancer, including thyroid adenocarcinomaand medullar carcinoma; and renal cancer including adenocarcinoma andWilms tumor. Viral proteins associated with tumors would be those fromthe classes of viruses noted above. Antigens characteristic of tumorsmay be proteins not usually expressed by a tumor precursor cell, or maybe a protein which is normally expressed in a tumor precursor cell, buthaving a mutation characteristic of a tumor. An antigen characteristicof a tumor may be a mutant variant of the normal protein-having analtered activity or subcellular distribution. Mutations of genes givingrise to tumor antigens, in addition to those specified above, may be inthe coding region, 5′ or 3′ noncoding regions, or introns of a gene, andmay be the result of point mutations frameshifts, deletions, additions,duplications, chromosomal rearrangements and the like. One of ordinaryskill in the art is familiar with the broad variety of alterations tonormal gene structure and expression which gives rise to tumor antigens.

Specific examples of tumor antigens include: proteins such asIg-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 ofmelanoma, Pmel-17 (gp 100) of melanoma, MART-1 (Melan-A) of melanoma,p15 protein of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 ofmelanoma, thyroid medullary, small cell lung cancer, colon and/orbronchial squamous cell cancer, BAGE of bladder, melanoma, breast, andsquamous-cell carcinoma, gp75 of melanoma, oncofetal antigen ofmelanoma; carbohydrate/lipids such as muci mucin of breast, pancreas,and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes suchas mutant p53 of carcinoma, mutant ras of colon cancer and HER21neuproto-onco-gene of breast carcinoma; viral products such as humanpapilloma virus proteins of squamous cell cancers of cervix andesophagus; and antigens (shown in parenthesis) from the followingtumors: acute lymphoblastic leukemia (etv6; aml1; cyclophilin b), glioma(E-cadherin; α-catenin; β-catenin; γ-catenin; p120ctn), bladder cancer(p21ras), billiary cancer (p21ras), breast cancer (MUC family; HER2/neu;c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras;HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectalassociated antigen (CRC)—C017-1A/GA733; APC), choriocarcinoma (CEA),epithelial cell-cancer (cyclophilin b), gastric cancer (HER2/neu;c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein),hodgkins lymphoma (lEBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1),lymphoid cell-derived leukemia (cyclophilin b), myeloma (MUC family;p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2),nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer cancer (MUCfamily; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen(PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA;HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu;c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-2), testicularcancer (NY-ESO-1), T cell leukemia (HTLV-1 epitopes), and melanoma(Melan-A/MART-1; cdc27; MAGE-3; p21ras; gp100^(Pmel)117). It is alsocontemplated that proteinaceous tumor antigens may be presented by HLAmolecules as specific peptides derived from the whole protein. Metabolicprocessing of proteins to yield antigenic peptides is well known in theart; for example see U.S. Pat. No. 5,342,774 (Boon et al.). and the oneson the lists previously.

Antigens may also include: C reactive protein; Coxsackie B1, B2, B3, B4,EI5, B6 proteins; Myelin basic protein; pancreatic beta-cell antigens;arthritis associated antigens (cartilage, aggrecan, type II collagen);AP-1; NF-kappaB; desmoglein (Dsg 1 or 3); and alzheimer's associatedantigens (prions, amyloid-beta protein), and/or any synthetic agent thatbinds to the T-cell receptor.

Further exemplary cancer, viral, and beta islet autoantigens aredescribed below in Tables 2, 3, and 4 respectively.

TABLE 2 Exemplary Cancer Antigens Protein MHC Peptide Position SEQ IDNO: MAGE-A1 HLA-A1 EADPTGHSY 161-169 1 HLA-Cw16 SAYGEPRKL 230-238 2MAGE-A3 HLA-A1 EVDPIGHLY 168-176 3 HLA-A2 FLWGPRALV 271-279 4 HLA-B44MEVDPIGHLY 167-176 5 MAGE-A6 HLA-Cw16 KlSGGPRISYPL 292-303 6 MAGEmelanoma ALSRKVAEL 7 AG BAGE HLA-Cw16 AARAVFLAL  2-10 8 GAGE-1,2HLA-Cw16 YRPRPRRY  9-16 9 RAGE HLA-B7 SPSSNRIRNT 11-20 10 GnT-V HLA-A2VLPDVFIRC 2-10/11 11 MUM-1 HLA-B44 EEKLIVVLF exon 12 2/intron EEKLSVVLF(wild type) 13 CDK4 HLA-A2 ACDPHSGHFV 23-32 14 ARDPHSGHFV (wild type) 15β-catenin HLA-A24 SYLDSGIHF 29-37 16 SYLDSGIHS (wild type) 17 TyrosinaseHLA-A2 MLLAVLYCL 1-9 18 HLA-A2 YMNGTMSQV 369-377 19 HLA-A2 YMDGTMSQV369-377 20 HLA-A24 AFLPWHRLF 206-214 21 HLA-B44 SEIWRDIDF 192-200 22HLA-B44 YEIWRDIDF 192-200 23 HLA-DR4 QNILLSNAPLGPQFP 56-70 24 HLA-DR4DYSYLQDSDPDSFQD 448-462 25 HLA-A2 ILTVILGVL 32-40 26 gp100^(Pmel117)HLA-A2 KTWGQYWQV 154-162 27 HLA-A2 ITDQVPFSV 209-217 28 HLA-A2 YLEPGPVTA280-288 29 HLA-A2 LLDGTATLRL 457-466 30 HLA-A2 VLYRYGSFSV 476-485 31PRAME HLA-A24 LYVDSLFFL 301-309 32 NY-ESO-1 HLA-A2 SLLMWITQCFL 157-16733 HLA-A2 SLLMWITQC 157-165 34 HLA-A2 QLSLLMWIT 155-163 35 c-erb2HLYQGCQVVPLTSIISAV 36 p53 264-272 LLGRNSFEV 37

TABLE 3 Exemplary Viral Antigens SEQ Protein MHC Peptide Position ID NO:Rubella E1 WVTPVIGSQARKCGL 276-290 38 RVIDPAAQ 412-419 39 Measles FHQALVIKLMPNITLL 40 Papilloma RLCVQSTHV 41 YVRDGNPYA E6 60-68  42GYNKPLCDLL E6 98-107 43 Influenza KGILGFVFTLTV 57-68 44 matrix influenzaHA EKYVKQNTLKLAT 307-319 45 Hepatitis B WLSLLVPFV 46 SAg FLGGTTVCL 47Hepatitis C YLVAYQATV 48 NS NS3 GLRDLAVAV 49 GYKVLVLNPSVAAT 1248-1261 50KLVALGINAV 1406-1415 51 Tetanus QYIKANSKFIGIYQL 830-843 52

TABLE 4 Exemplary Beta Islet Cell Autoantigens: Protein Peptide PositionSEQ ID NO: glutamic acid TYELAPVFVLLEYVT 206-220 53 decarboxylase 65LKKMRFIIGWPGGSG 221-235 54 KKGAAAIGIGTDSVI 286-300 55 PLOCSALLVREEGLM401-415 56 WLMWRAKGTTGFEAH 456-470 57 tyrosine VIVMLTPLVEDGVKQC 805-82058 phosphatase IA-2

One or more antigens can be used at the same time for incubation in theforegoing culture system. Preferably, the lymphoreticular stromal cellsare thymic stromal cells and of murine origin when the hematopoieticprogenitor cells being expanded are human. Therefore, large numbers ofantigen-specific mature T and immature T cells may be obtained in ashort period of time that were never before realized using existing artmethodologies. The present invention thus becomes useful in a wide rangeof applications, including pre-exposure vaccination of individuals within vitro primed T cells, treatment of cancer patients usingtumor-targeted T cell immunotherapy, treatment of bone marrow transplantpatients (for whom opportunistic infections, such as CMV, areproblematic and yet amenable to treatment with targeted T cells such asCMV-targeted cytotoxic lymphocytes), enhancement of conventionalvaccination efficacy through T cell adjuvant therapy, treatment ofoutbreaks of emergent or re-emergent pathogens, etc. The antigenpresenting cells include cells such as dendritic cells,monocytes/macrophages, Langerhans cells, Kupfer cells, microglia,alveolar macrophages and B cells, and methods for their isolation arewell known in the art. The antigen presenting cells may also be derivedfrom hematopoietic progenitor cells in vitro.

Immunological tolerance refers to the inhibition of a subject's abilityto mount an immune response, e.g., to a donor antigen, which wouldotherwise occur in response to the introduction of a non-self antigeninto the subject. Tolerance can involve humoral, cellular, or bothhumoral and cellular responses. Thymic education results in thegeneration of T cells capable of responding to a myriad of foreignantigens in the context of self-MHC, but not self-antigens alone. Thisis achieved primarily by a systematic rescue of appropriate thymocytesfrom programmed cell death, based on a theme of self-restriction, andthe release of these cells into the periphery to serve as self-tolerantT cells.

Methods of the invention are useful, inter alia, for generating largenumbers of lymphoid tissue-specific cells that are educated towardspecific cells/antigens, and are therefore tolerant toward the specificcells/antigens. For example, according to the invention and in thecontext of transplantation, a recepient's hematopoietic progenitor cellsare co-cultured, in one instance, with a donor's lymphoreticular stromaand/or a donor's antigen presenting cells. The lymphoid tissue-specificcells that are generated from such co-culture are educated towarddonor's cells/antigens and are therefore tolerant, thus increasing thelikelihood of a successful transplantation of an donor organ into arecipient (host). Various other permutations are therefore contemplatedby the invention (see summary), where non-autologous cells (e.g., donorcells) can be co-cultured in the presence of other non-autologous orautologous (e.g., recipient) cells. Non-autologous cells, as discussedearlier, are cells that originate from a different subject (see earlierdiscussion). They can be from a single source (e.g., hematopoieticprogenitor cells and lymphoreticular cells from one subject), or frommultiple sources (e.g., two different subjects -hematopoietic progenitorcells from one subject and lymphoreticular cells from another subject).

Exemplary permutations include:

Prog. cell Non-autologous* progenitor cells Autologous (Recipient)progenitor cells source Stroma Non-autologous Recipient stromaNon-autologous Recipient stroma source stroma stroma APC Non- RecipientNon- Recipient Non- Recipient Non- Recipient source autologous APCsautologous APCs autologous APCs autologous APCs APCs APCs APCs APCs *canbe from one source or multiple sources

Self-tolerance can be established in vitro under conditions known in theart that include coculturing CD34⁺ T progenitors derived from a donor(A), in the presence of thymic stroma from another individual (B).Briefly, thymic stroma is established from freshly isolated thymictissue that is digested into a single cell suspension using acollagenase (20 μg/ml, Sigma Chemical Co.). Thymic stromal cultures areestablished by plating the cell suspension in 24 well plates at aconcentration of 4×10⁶ viable cells per well in a volume of 2 ml R10(RPMI plus 10%FCS). Cultures are incubated in a standard humidifiedtissue culture incubator at 37° C. with 5% CO₂. After one to two days,non-adherent cells are removed by washing three times with R10. Thestroma requires an additional 7-10 days to become confluent. The stromais maintained in R10 which is changed at least twice per week. After7-10 days in culture, CD34⁺ cells in R10 are added to the stroma at aconcentration of 1-3×10⁵ cells per well. Cultures are fed bi-weeklyusing partial medium exchanges with R10 with no exogenous cytokinesadded to these cultures. After 14-21 days, the non-adherent cells areremoved from the cultures. The remaining, attached cells areself-tolerant T cells that have developed in vitro.

Methods for determining if tolerance has been established in vitro arealso known to a person of ordinary skill in the art, and involvemeasurement of a proliferative response to: self (A), as well as to thethymus donor (B), and a third party (C), peripheral blood mononuclearcells (PBMCs). Briefly, PBMCs from A, B and C are prepared by Ficollgradient centrifugation.

1×10⁵ responder cells (in vitro generated T cells from A) are plated outin multiple replicates in a 96 well plate. Stimulator cells (PBMCs fromA, B and C) are irradiated (3000 Rads) and added in 12 replicates at1×10⁵ cells per well. Con-A (5 μg/ml) is used as a positive control.After 4 days 1 μCi of ³H-Thymidine is added to each well, and the platesharvested 18-24 hours later. If tolerance has been established, the invitro generated T cells will respond and proliferate when mixed with anunrelated third party (C), but do not proliferate when mixed with PBMCsfrom self (A) or the thymic donor (B).

According to another aspect of the invention, a method for inducing Tcell anergy, in vitro, is provided. Induction of T cell anergy involvesco-culturing the hematopoietic progenitor cells and the lymphoreticularstromal cells in one of the foregoing matrices, in the presence ofantigen under conditions sufficient to induce the formation of T cellsand/or T cell progenitors and to inhibit activation of the formed Tcells and/or T cell progenitors.

Anergy is defined as an unresponsive state of T cells (that is they failto produce IL-2 on restimulation, or proliferate whenrestimulated)(Zamoyska R, Curr Opin Immunol, 1998, 10(1):82-87; VanParijs L, et al., Science, 1998, 280(5361):243-248; Schwartz R H, CurrOpin Immunol, 1997, 9(3):351-357; Immunol Rev, 1993, 133:151-76). Anergymay, however, be irreversible. Anergy may be induced viaantigen-specific T cell stimulation in the absence of co-stimulation(one signal vs. two signal hypothesis). Alternatively peptides of lowaffinity or very high concentrations of peptide even in the presence ofco-stimulation can induce anergy. Anergy can be induced in vitro byculturing T cells in the absence of antigen presenting cells (B cells,macrophages or dendritic cells). These T cells are then exposed toantigen for example tetanus toxoid (e.g., 0.1-1.0 μM). An aliquot of theT cells is used to present antigen. This constitutes antigenpresentation without co-stimulation and will induce anergy (Nelson A, etal., In Immuno, 1998, 10(9):1335-46). Alternatively T cells can becocultured with APCs, in the context of very high (10-100 μM) or verylow (0.01-0.05 μM) tetanus toxoid, which will induce a state ofunresponsiveness.

Anergy can be measured by taking the T cells described above, andrestimulating them with antigen (e.g., 0.1-1.0 μM tetanus toxoid) in thepresence of APCs. If the cells are anergic they will not respond toantigen at an appropriate concentration in the context of APCs. Anergyis measured by culturing the cells as such for 3-5 days and measuringproliferation or the lack thereof as follows. Briefly APCs are platedout in multiple replicates in a 96 well plate, after irradiation (3000Rads). These cells are pulsed with antigen (e.g., 0.1-1.01 μM) for 2hours , and then T cells are added in 12 replicates at 1×10⁵/cells perwell. Con-A (5 μg/ml) is used as a positive control. After 4 days 1 μCiof ³H-Thymidine is added to each well, and the cells are harvested 18-24hours later. If the cells are anergic they will not proliferate inresponse to antigen stimulation. Alternatively, the production of IL-2can be measured in the supernatants of the cultures described above.Supernatants are collected daily and IL-2 production is measured using acommercial ELISA assay. An additional approach includes flow cytometrybased staining specific for intracellular expression of the cytokinesIL-2, γIFN and TNFα using antibodies specific to the human forms ofthese factors (Becton Dickinson). Further, semiquantitative RT-PCR ofmRNA for these factors can also be used.

According to another aspect of the invention, a method for identifyingan agent suspected of affecting hematopoietic cell development isprovided. The method involves introducing an amount of hematopoieticprogenitor cells and an amount of lymphoreticular stromal cells into aporous, solid matrix of the invention, and co-culturing in a testco-culture the hematopoietic progenitor cells and the lymphoreticularstromal cells in the presence of at least one candidate agent suspectedof affecting hematopoietic cell development. By “hematopoictic celldevelopment” it is meant to include hematopoietic progenitor cellmaintenance, expansion, differentiation, and/or cell-death apoptosis(programmed cell-death). “Maintenance” includes the hematopoieticprogenitor cell's ability to maintain its pluripotentiality. “Expansion”includes the hematopoietic progenitor cell's ability to divide and grow,and “differentiation” includes the hematopoietic progenitor cell'sability to differentiate toward a specific cell lineage. “Cell-death”also includes programmed cell-death (apoptosis). By “affecting”hematopoietic cell development it is therefore meant to include effectson hematopoietic progenitor cell maintenance, expansion,differentiation, and/or cell-death. Such effect (or influence) can beeither positive or negative/inhibitory in nature. For example, apositive effect would be maintenance of pluripotentiality of theprogenitor cells, and/or increase in the number of the pluripotentialprogenitor cells. A negative effect would lead into the differentiationof the progenitor cells and loss of pluripotentiality, or evenprogenitor cell-death. A negative effect on a particular cell populationmay also have a positive effect on a different cell population. Forexample, an inhibitory effect on a B cell lineage may result in apositive effect on, for example, a T cell lineage. The agent suspectedof affecting hematopoietic cell development may be administered in theform of a transfected nucleic acid into the lymphoreticular stromalcells as well as being added straight into the media.

To determine whether the at least one candidate agent affectshematopoietic cell development in the test co-culture, the phenotypeand/or genotype (as well as the numbers) of the hematopoietic cellsgenerated in the test co-culture is compared to the phenotype and/orgenotype (and numbers) of hematopoietic cells generated in a controlco-culture. The control co-culture is performed under identicalconditions to the test co-culture (i.e., identical initial numbers andtypes of both hematopoietic progenitor cells and lymphoreticular stromalcells, in an identical matrix, identical culture media, etc.), but withthe exception that the at least one candidate agent suspected ofaffecting cell hematopoietic cell development is omitted from thecontrol co-culture. Methods for determining the phenotype and/orgenotype of hematopoietic cells are well known in the art, and a fewexamples can be found throughout this application.

In yet another aspect of the invention, a method for isolating from acell culture an agent suspected of affecting hematopoietic celldevelopment is also provided. The method involves introducing an amountof hematopoietic progenitor cells and an amount of lymphoreticularstromal cells into a porous, solid matrix of the invention, co-culturingthe hematopoietic progenitor cells and the lymphoreticular stromal cellsand obtaining a test- supernatant (or a fraction thereof) from theco-culture. The test-supernatant (or a fraction thereof) is thencompared to a control-supernatant (or a fraction thereof). By“comparing” it is meant that a profile of agents (suspected of affectinghematopoietic cell development) present in the test-supernatant andsecreted from the cells of the co-culture, is compared to a similarprofile of agents present in the control-supernatant and secreted fromthe cells of a control culture or co-culture. Methods of obtaining suchprofiles of secreted agents are well known in the art and includetwo-dimensional (2-D) gel electrophoresis. Other methods also includevarious types of HPLC, thin layer chromatography.

A “control culture or co-culture” may involve the culture ofhematopoietic progenitor cellsin a parallel culture system known in theart (e.g. U.S. Pat. No. 5,677,139 by Johnson et al.), in order to obtaina result that correlates (i.e. approximates) to the result establishedin the co-culture system of the invention. For example, a testco-culture according to the invention that involves the co-culture ofhuman hematopoietic progenitor cells and lymphoreticular stromal cellsfrom a mouse thymus, gives rise to a diverse (a variety of sub-types)population of human lymphoid cells committed to the T cell lineage. Thetest-supernatant obtained from such co-culture is then compared to acontrol-supernatant obtained from a culture of human hematopoieticprogenitor cells in a parallel system of the prior art (as describedabove) that also gives rise to a population of human lymphoid cellscommitted to the T cell lineage. Other examples of control cultures orco-cultures may include the co-culture of hematopoietic progenitor cellswith lymphoreticular stromal cells of different tissue origin to theones used in the test co-culture in the matrix of the invention.Additionally, the tissue may or may not be of lymphoid origin. A personof ordinary skill in the art would be able to easily choose andestablish such control cultures or co-cultures. Once the profiles ofagents suspected of affecting hematopoietic cell development areobtained, a subfraction of the test-supernatant that contains an agentsuspected of affecting hematopoietic cell development that appears to bedifferent or absent from the control-supernatant, can then be isolatedand further characterized. For example, a candidate agent that appearsto be migrating differently in a 2-D gel electrophoresis blot of thetest-supernatant can be purified and further characterized using methodssuch as protein sequencing and mass spectrometry. Agents that appear inthe 2-D gel electrophoresis blot but are absent from the blot of thetest-supernatant are also suspect of affecting hematopoietic celldevelopment and can be further purified.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES

Experimental Procedures

Isolation of Human CD34⁺ cells

Five to ten milliliters of venous umbilical cord blood (UCB) wasextracted using a heparinized syringe prior to the severing of theumbilical cord during a Caeserean section delivery of a human embryo.After the umbilical cord was severed and the infant delivered, theplacenta was removed by clamping the umbilical vein proximally andsevering distally to the placenta. Immediately after the placenta wasremoved the umbilical vein was unclamped and the blood contained in theplacenta drained into an appropriate heparinized container. Beforeprocessing, the cord and placenta blood was mixed together. Afterextraction the cord/placenta blood was diluted 2:1 with washing media(RPMI 1640, 10 IU/ml penicillin, 10 μg/ml streptomycin, 1 mML-glutamine). The sample(s) were then underlayed with a volume ofFicoll-Hypaque (1.077 g/ml) equal to half of the diluted sample volumeso that a distinct sample/Ficoll interface formed. After centrifugationfor 45 minutes at 400×g the interface containing mononuclear cells wasremoved. The cells were then washed by resuspending in culture mediumand centrifuging for 10 minutes at 400×g. The resulting pellet wasresuspended in 6 ml of ammonium chloride lysing buffer (0.15 M NH₄Cl,1.0 mM KHCO₃, 0.1M Na₂EDTA) for 3 minutes to lyse any remainingerythrocytes. The suspension was then diluted with media and washedtwice more. After the final wash the cells were resuspended in 1-2 mlmedia and the number of viable cells was determined by trypan blueexclusion.

Human CD34⁺ progenitor cells were also prepared from disaggregated humanfetal thymus obtained from 16-22 week old abortuses. For dissagregationprocedures see below under Mouse Thymic Stroma.

Cells expressing the surface antigen CD34 were isolated using the DynalCD34 Progenitor Cell Selection System (Dynal, Lake Success, N.Y.) or theMiniMACS system (Miltenyi Biotec, Bergisisch Gladbach, Germany). Themononuclear cells isolated from UCB (or bone marrow) were suspended inisolation buffer (PBS, 2% heat inactivated fetal bovine serum, 10 IU/mlpenicillin, 10 μg/ml streptomycin) at a concentration of 2.5×10⁷cells/ml. The suspension was then added to magnetic anti-human CD34beads (Dynal M-450 CD34) in a ratio of 4.0×10⁷ beads per ml ofsuspension, in a round bottom tube. (Dynabeads M-450 CD34 aresuperparamagnetic beads bound to monoclonal antibody specific for CD34).The mixture was vortexed gently and incubated at 4° C. for 45 minuteswith gentle tilt rotation using a Dynal Sample Mixer. After incubationthe bead/cell mixture was resuspended in a larger volume of isolationbuffer and placed in a magnetic separation device for 2 minutes to allowthe cell/bead complexes to accumulate to the tube wall. While stillexposed to the agent, the suspension containing the cells not bound tothe magnetic beads was aspirated. The cell/bead complexes were washedthree more times in this manner, pooling the suspensions containing theCD34 negative cells into the same tube. The tube containing the releasedcells (CD34−) was then placed on the magnetic separator to remove anyremaining beads and this supernatant was transferred to a new conicaltube. All CD34⁺ cells attached to beads were washed twice in a minimumof 10 ml of isolation buffer with centrifugation at 2000 rpm for 8 min.Cells bound to magnetic beads were then resuspended in 100 μl ofisolation buffer per 4×10⁷ beads used, with a minimum volume of 100 μl.The CD34 positive cells were then detached from the beads by adding anequal volume of an anti-idiotype antibody (DETACHaBEAD CD34, Dynal),vortexing, and gently mixing at room temperature using a Dynal SampleMixer for one hour. The cells were isolated from the cell/beadsuspension by adding isolation buffer and placing the tube in themagnetic separation device for 2 minutes. After the beads migrated tothe tube wall, the supernatant containing the CD34 positive cells wastransferred to a new tube. The beads were washed three more times withthe suspensions containing the released cells pooled into the same tube.The tube containing the released CD34⁺ cells was then placed on themagnetic separator to remove any remaining beads, and the supernatantwas transferred to a new conical tube. The cells were washed twice in aminimum of 10 ml of isolation buffer with centrifugation at 2000 rpm for10 minutes.

Alternatively, human bone marrow was obtained by posterior iliac crestaspiration from healthy adult volunteers in accordance withinstitutional review board guidelines and after giving informed consent.10-15 mL of human bone marrow was collected in an heparinized sterilesyringe, transported at room temperature and used within 6 hours. Bonemarrow was diluted in a 5-times volume of PBS and the mononuclear cells(MNCs) separated by density gradient centrifugation over a column ofFicoll-Paque (Pharmacia Biotech Inc., Piscataway, N.J.). MNCs thusobtained were washed twice in 10 mL PBS, and the remaining erythrocytesremoved by lysis with ACK Lysing Buffer (Bio Whittaker, Walkersville,Md.).

In order to select a more immature phenotype of progenitor cell withinthe CD34⁺ population, we elected to use an immunomagnetic bead selectionsystem employing an antibody to the novel stem cell antigen, AC133.AC133 is a 5-transmembrane cell surface antigen expressed on 20-60% ofhuman CD34⁺ cells, including the CD38^(neg/dim) subset (representing thenon-lineage-committed precursors) but is not expressed on matureleukocytes (Yin AH, et al., Blood, 1997. 90:5002-12; Nfiraglia S, etal., Blood, 1997, 90:5013-21; Buhring H J, et al., Ann N Y Acad Sci,1999, 872: 25, discussion 38-9). Although a small number of mature CD2⁺T-cells were transferred into our co-cultures with the AC133⁺progenitors we do not believe that the T-cells generated in this systemare derived from either CD2⁺ mature lymphocytes or CD2⁺lymphoid-committed precursors. We, and others (Fisher A G, et al., IntImmunol, 1990, 2:571-8), have observed that the deliberate introductionof mature human T-cells into the co-cultures does not result inincreased numbers of T-cells or their precursors. The AC133⁺ MNCfraction was isolated by immunomagnetic bead selection using an AC133Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, Calif.) according tothe manufacturer's protocols.

Mouse Thymic Stroma

Thymi were obtained from freshly sacrificed 6 week-old B6(BALB/C) mice.Thymi were physically disaggregated with surgical scissors in order toproduce a cell suspension which also contained fragments of thymictissue less than 0.5 mm³ in size. The cell suspension containing thymicfragments was plated onto 0.5 cm×0.5 cm×0.2 cm pieces of Cellfoam (80ppi), placed in each well of a 24 well plate. Each well contained atleast 5×10⁶ cells and 4 fragments of fetal thymus per Cellfoam block andthe cells were cultured in fully supplemented IMDM. The medium in thymiccultures was changed initially at 48 hours post establishment of theculture and at three day intervals there after. On average 80% confluentthymic stromal monolayers were established on Cellfoam between 10 and 14days. At 10 to 14 days of culture the Cellfoam blocks each containing asub-confluent layer of thymic stroma were removed from the 24 well plateand placed in the wells of a new 24 well plate, and co-cultured withCD34⁺ cells.

Human CD34⁺/Murine Thymic Stroma Co-Culture Conditions

Five thousand CD34⁺ cells derived from UCB or human bone marrow werethen plated onto the irradiated murine thymic stroma. In the case whereCellfoam was used, CD34⁺ cells were plated directly onto the Cellfoamitself in the well of the 24 well tissue culture dish. Medium inco-cultures was changed every three days and was not supplemented withexogenous cytokines. Cells generated from the CD34⁺ cells were harvestedat 7 days post establishment of the co-culture and flow-cytometric andfunctional studies were performed on the derived cells.

Assessment of Immunophenotype and Function of Cells Derived from theCo-cultures

Adherent cells were harvested with a non-trypsin isolation solution(Cell Dissociation Solution, Sigma, St. Louis, Mo.) to minimizealteration of surface staining characteristics. To recover adherentcells from Cellfoam, units were washed twice by immersion into PBS,saturated by brief vortexing in an excess of Cell Dissociation Solution,incubated for 20 minutes at 37° C., and centrifuged at 1500 rpm for 10minutes.

Cells were harvested by gentle aspiration and washed twice in PBS.Harvested cells were counted and assessed for viability by trypan blueexclusion. After counting, cells were stained in a final volume of 100μL with 2% mouse serum (Dako, Carpentiera, Calif.) and the followingfluorochrome-conjugated antibodies: TCRαβ, TCRγδ, CD2, CD3, CD4, CD8,CD14, CD33 and CD34(Becton Dickinson, San Jose, Calif.). Conjugatedisotype control antibodies for all four fluorochromes (FITC, PE,Peridinin chlorophyll protein (PerCP), and Allophycocyanin (APQ wereused for each culture. Stained samples were washed three times with PBS,fixed with 1% paraformaldehyde, and analyzed with a FACScalibur flowcytometer (Becton Dickinson). Appropriate controls included matchedisotype antibodies to establish positive and negative quadrants, as wellas appropriate single color stains to establish compensation. For eachsample, at least 10,000 list mode events were collected. Anti-CD3 andanti-CD14 were utilized to detect contaminating T-cells and monocytes inthe CD34+ selected MC subpopulation.

Human leukocytes were distinguishable from murine cells onimmunophenotypic analysis by gating on the CD45⁺ population. After 14days in co-culture, >70% of CD45⁺ cells coexpressed CD3, CD4, and/orCD8. It was possible to track the sequential differentiation ofT-lymphoid precursors in this system over 2 weeks (FIG. 1). CD34⁺progenitors added into co-culture with a murine thymic stroma cells, ona three-dimensional matrix (Cellfoam). Non-adherent cells were harvested7, 14 and 21 days after establishment of the co-cultures and theirimmunophenotype determined by FACS analysis. The data in panel (a)demonstrate the acquisition of CD2 and the down-regulation of thehematopoietic progenitor cell marker, CD34. Acquisiton of cell surfaceCD4 and CD8 markers occurred after 14 days in coculture; (b): discretepopulations of SP CD4⁺ and SP CD8⁺ are demonstrated including their DPCD4⁺CD8⁺ precursors. Acquisition of CD4 at day 14 was associated withacquisiotion of CD3; (c and d): all CD4⁺ cells co-expressed CD3. CD3 wasco-expressed with the majority of CD8⁺ cells; those cells which wereCD3⁻CD8⁺ were found to express TCRγδ. TCRαβ was expressed by 78% of CD3⁺cells although a smaller population (20%) of CD3⁺ cells expressing TCRγδwas also detectable (6% CD3⁺CD8⁺ TCRγδ, 14% CD3⁺CD8⁺TCRγδ).

T Cell Function

T cell function was assessed by determining CD69 expression in responseto mitogens and ³H-Thymidine uptake in response to the mitogen Con-A. Tcells generated in the co-culture system were also examined for theirinfectability by HIV-1 and their transducability by the MFG murineretroviral vector. T cells generated from the co-culture showed expectedhigh levels of ³H-Thymidine uptake (10×control unresponsive cells) inresponse to the mitogen ConA and a four-fold increase in the expressionof the activation marker CD69 as determined by flow cytometry.

HIV-1 Challenge of T-cells Generated from HPC/thymic Stromal Co-cultures

T-cells generated from HPCs were challenged with T-cell tropic isolateHIV_(IIIB) at a multiplicity of infection of 1. Titered stocks of HIV-1were generated by standard means well known in the art. Samples ofculture supernatant were removed from cultures at 3, 6, 9, 14 and 28days post HIV challenge for HIV-1 p24 antigen estimation by ELISA(Coulter, Miami, Fla.). Secondly, sorted CD4⁺ T cells generated fromco-cultures of BM HPCs with thymic stroma were challenged withHIV_(IIIB) at a multiplicity of infection of 1. Cell viability was alsodetermined following challenge of monocytes and T cells with HIV-1 usingtrypan blue exclusion. T cells generated from HPCs were infectable withHIV-1 and produced up to 0.69 ng/ml of HIV-1 p24 by day 10 of culture.Both unsorted and sorted T cells generated form the co-cultures of HPCson murine thymic stroma on Cellfoam and exposed to heat inactivatedHIV_(IIIB), produced undetectable levels of HIV-1 p24. The viability ofT cells also declined significantly following exposure to infectionsHIV-1. The levels of HIV-1 p24 antigen production in T cells generatedform the Cellfoam co-culture system was similar to levels of HIV-1 p24production from human activated peripheral blood T cells.

Transduction of T Cells Generated from HPC/thymic Stromal Co-cultureswith an Amphotropic Murine Retroviral Vector

T cells generated from HPCs and expanded in IL-2 and PHA were exposed tothe murine retrovirus based vector, MFG, encoding the intranuclearlocalized enzyme β-galactosidase at an M.O.I. of 10 on three occasionsover the period of 72 hours. Titred retroviral vector was generated bystandard means from a human based FLYA4 packaging cell-line. T cellswere also exposed to heat inactivated MFG. Transduced cells wereharvested from cultures at 7 days following retroviral exposure andstained by standard methods for the expression of the beta-galactosidasetransgene. Transduction efficiencies of between 12 and 26% were observedin T cells generated from co-cultures of HPCs with murine thymic stromagrown on Cellfoam. No β-galactosidase activity was detectable in T cellsexposed to the heat inactivated retroviral vector. The transductionefficiency of human T cells generated from the Cellfoam co-culturesystem is similar to that seen in activated peripheral blood T cells.

mRNA Extraction and cDNA Synthesis

Generated cells were also lysed and RNA was prepared from the cells forRNA PCR in order the determine T cell receptor gene expression.Messenger RNA was extracted from cells grown on a thymic monolayer. Theextraction was performed using guanidinium thiocyanate and oligo-dT spuncolumns (QuickPrep Micro mRNA Purification Kit; Pharmacia, Piscataway,N.J.) according to the manufacturer's instructions. mRNA samples werestored at −70° C. The first strand cDNA was synthesized in a 40 μl finalvolume, using approximately 2 μg of mRNA, 1 μg of random primer, and6.25 units of AMV reverse transciptase (GIBCO/BRL). Samples wereincubated for 10 minutes at room temperature, 1 hour at 42° C., 5minutes at 95° C., and 5 minutes at 4° C. RT-PCR for a number oflymphoid-specific genes (including RAG-2) was performed using reversetranscription using random primers and Moloney MuLV reversetranscriptase (GIBCO-BRL, Grand Island, N.Y.). cDNAs were amplifiedusing gene-specific primers, e.g., for the human RAG-2 gene which isexpressed transiently only by cells undergoing lymphocytedifferentiation, Vβ gene expression, and the like. PCR amplificationwere performed in a GeneAmp 9600 thermal cycler (Perkin Elmer, Norwalk,Conn.) using conditions well known in the art.

Example 1

Viability, Immunophenotype and Function of Human Cells Generated inCo-Culture Systems

The numbers of viable cells generated in the co-culture system and theirimmunophenotype are shown in Table 5. Maximal human T cell proliferationwas seen when human fetal thymic CD34+ cells and UCB CD34+ cells wereco-cultured with murine fetal thymic stroma grown on Cellfoam. Datagenerated from a direct comparison of co-culture of CD34+ cells onmurine thymic stroma on cell foam versus co-culture of CD34+ cells onmurine stroma grown as a simple monolayer are also shown in Table 5.

T cells generated in the co-culture system were also shown to beinjectable by T-tropic HIV-1_(IIIB) and these cells were alsotransductible at a transduction efficiency of 12-22% (n=3) with MFGvector.

Example 2

Maintenance of Immature Progenitor Cells

According to the invention, it has also been discovered that Cellfoamcultures of thymic stromal cells are able to induce T celldifferentiation of CD34⁺ progenitors and yet preserve a fraction ofCD34⁺ cells. Primate CD34⁺ progenitors were cultured on either human orswine thymus that had been established on Cellfoam tissue scaffolds.After 14-21 days, CD3+CD4+CD8+ triple positive cells and CD3+CD4+ andCD3+CD8+ double positive cells are reliably recovered. In addition, theCD3-cell fraction was found to contain CD34⁺ progenitor cells after14-21 days. These CD34⁺ cells not only were CD3−, but many were alsoCD2+. This demonstrates that thymus cultures in Cellfoam tissuescaffolds can support T cell differentiation while simultaneouslypreserving the long-lived CD34⁺ progenitor cell population. As will beevident to those skilled in the art, this surprising finding indicatesthat ongoing differentiation of T progeny while maintaining immatureprogenitor cells is possible in Cellfoam.

Example 3

T Cell Function (Proliferation/Anergy) Assays

T cell function is evaluated by the proliferative potential to specificand non-specific antigens using standard assays. Specifically, the assayassesses the response of T cell receptor (TCR) mediated proliferationusing anti-CD3 antibodies (Becton Dickinson) as well as baselinenon-specific proliferation using concavalin A (Con-A). Briefly, T cellsare washed and resuspended in RPMI with 10% FCS at a concentration of10⁶ cells/ml. 100 μl (10⁵ cells) are added to each well of a 96 wellplate. Cells are stimulated with either Con-A (5 μg/ml) (non-specificresponse) or monoclonal antibodies to CD3 in the presence of IL-2(20units/ml) and irradiated mononuclear cells (MCs) (10⁵ cells/well in 100ml of RPMI with 10% FCS). Purified goat anti-mouse F(ab′)₂ fragments(Kirkegard and Perry Laboratories, Gaithersberg, Md.) are used as acrosslinking agent for the experimental conditions where monoclonalantibodies to CD3 are used. Wells are pretreated with 1.25 μg/ml of goatanti-mouse antibody for 45 minutes at 37° C. and washed three timesprior to the addition of monoclonal antibodies to CD3 and CD28. Controlsincluded T cells alone, T cells plus irradiated MCs, and T cells plusmitogenic stimuli without IL-2 or irradiated MC. After 7 days in cultureat 37° C., cell proliferation are assessed using either radio-activeassays or commercially available non-radioactive, ELISA based assays(e.g. Promega). Cells are co-cultured for 5-7 days to induceproliferation of the T cells (the stimulator cells are also irradiatedand thus non-proliferative). Stimulator cells alone serve as controls.

An additional approach to testing T cell function uses flow cytometrybased staining for intracellular expression of the cytokines IL-2γIFNand TNFα using antibodies specific to the human forms of these factors(Becton Dickinson). These cytokines are produced in the T progeny in theantigen specific in vitro proliferation assays. This allows low leveldetection of human cells among a high proportion of mouse cells,selectively highlighting the human progeny and excluding the mousecells. Further, semiquantitative RT-PCR of mRNA for these factors canalso be used.

In one particular example, for instance, cells removed from co-cultureafter 14 days showed pronounced proliferation when placed in liquidculture with complete medium and IL-2(10 IU/mL) and phytohemagglutinin(PHA; 2 μg/mL). After a further 7 days in culture there was a 45-foldincrease in cell number: >90% were CD3⁺CD4⁺ TCRαβ⁺; 3% CD3⁺CD8⁺ TCRαβ⁺and 3% CD3⁺CD8⁺CD4⁺ TCRαβ⁺. No cells expressing TCRγδ were detected.

TABLE 5 USE OF THYMIC STROMA/HUMAN FETAL CD34+ CELL CO-CULTURE SYSTEMStandard Protocol Fetal Human 6 Week Murine Neonatal swine Fetal Human 6Week Murine Neonatal Swine n = 3 Human Thymic stroma Thymic stromaThymic stroma Thymic stroma Thymic stroma Thymic stroma UCB CD34+ n = 3n = 3 n = 3 n = 3 n = 3 n = 3 Human fetal Cell foam Cell foam Cell foamMonolayer Monolayer Monolayer thymic stroma Thy CD34+ Thy CD34+ ThyCD34+ Thy CD34+ Thy CD34+ Thy CD34+ d14 d28 d7 d7 d7 d7 d7 d7 CD4 2.83.8 81.54 85.8 67.8 88.2 81.1 69.2 CD3 1.5 1.1 12.75 87.1 80.7 13.1 90.179.2 CD4/CD3 4.2 1.4 9.83 75.2 64.65 10.6 68.1 66.3 CD8/CD3 2.1 2.8 0.1374.5 28.8 0.46 71.9 28.3 CD4/CD8 1.8 1 0.37 79.5 25.4 0.89 89.1 22.9 CD24.1 12.5 ND 23.2 50.7 ND 20.4 48.1 CD14 33.4 16.1 59.6 0.17 6.8 63.10.81 4.6 CD33 48.2 20.2 90.61 0.33 ND 84.1 0.59 ND CD2/CD14 1.6 7.3 ND0.85 ND ND 0.26 ND CD2/CD33 4.8 12.8 ND 3.76 ND ND 4.12 ND CD33/CD14 8.460.73 0.13 ND 56.13 0.11 ND Viable Cell Count t₀ = 5,000 195,000 210,00098,000 1,800,000 220,000 15,000 79,000 51,000

Example 4

T Cell Lymphopoiesis Assay

AC133⁺ progenitor cells were added to the murine thymic stromal culturesat cell densities of either 1×10⁵, 1×10⁴, or 1×10³ cells per well andcultured for an additional two weeks at 37° C. in a 5% CO₂ humidifiedatmosphere. Medium in the co-cultures was changed every 4 days and wasnot supplemented with exogenous cytokines. Cells generated from theprecursors were harvested 7 and 14 days after establishment of theco-cultures.

The selected AC133⁺ cells represented a highly purified progenitor cellpopulation. Immunophenotypic analysis showed that >98% were CD34⁺; noneco-expressed surface CD3, CD4 or CD8. A small number of contaminatingCD2⁺ cells were detectable by flow cytometry within the AC133+ selectedpopulation: this comprised only 0.57%±0.29% (mean±SEM; n=6) of the cellsobtained from the selection process.

Example 5

Determination of Optium Matrix Size and Input Cell Number

Having tested matrices of differing dimensions we have determined thatthe optimal sized matrix for use in this system measures 10 mmdiameter×1 mm in depth. Similarly, input cell density appears criticalfor optimum T-cell generation: no lymphocytes were generated using aninput cell density of less than 1×10⁴ cells per well. However, using10×1 mm matrices and input cell densities of 1×10⁴ or 1×10⁵, we wereable to generate large numbers of human cells, 71.21%±9.87% (mean SEM;n=7) of which were CD3⁺, after 14 days in co-culture.

Example 6

Intra- and Inter-Sample Variability in Numbers of T-Cells Generated

In order to determine the variation of T-cell output within the a givensource of progenitors, multiple co-cultures were established using asingle source of AC133⁺ cells at fixed cell densities (1×10⁴ cells perwell) on separate 10×1 mm matrices of murine thymic stroma. Theintrasample variation of T lymphocytes generated was analyzed by cellcount using trypan blue exclusion, and by immunophenotypic analysis.Human cells were distinguished by surface expression of CD45. After 7days in co-culture, the number of mature T-cells detected was extremelylow: CD3⁺ cells represented 2.02%±0.87% (mean±standard error) of theCD45⁺-gated population, CD3⁺CD4⁺ T-cells accounted for 1.0%±0.52% of thegated population and CD3⁺CD8⁺ 0.58%±0.1% of the same gated population.However, after 14 days, the numbers of T-cells were significantlyhigher: the proportion of CD3⁺ cells rose to 62.16%±4.53%; and thepercentages of CD3⁺CD4⁺ and CD3⁺CD8⁺ were 42.7%±2.87% and 22.39%±1.29%respectively. These data are represented graphically in FIG. 2.

The intersample variation was calculated by comparing the number ofT-cells generated from separate sources of CD34⁺ progenitors. In eachcase a fixed number of cells (1×10⁴ cells per well) had been introducedinto co-culture. Immunophenotypic analysis of cells generated after 7days in co-culture showed that, of the CD45 gated population,1.57%±⁻0.97% of cells expressed CD3; 2.27%±2.70% co-expressed CD3 andCD4; and 0.46%±0.23% expressed both CD3 and CD8. After 14 days, theimmunophenotype of the cells harvested revealed that 71.21%±9.87% wereCD3⁺; 37.44%±8.44% were CD3⁺CD4⁺, and 38.06%±19.13% were CD3⁺CD8⁺ asshown in FIG. 3.

These data demonstrate a high level of reproducibility within the systemthat suggests its potential in comparative analyses of inputpopulations.

Example 7

Analysis for TCR Excision Circles (TREC)

The TCR Vδ locus lies between the TCR Vα and TCR Jα segments. In orderto complete TCRα VD-J rearrangement, the TCR Vδ segment is excised: the3′ and 5′ ends of the gene unite to form an extra-chromosomal circle ofDNA termed a TCR excision circle (TREC) (Berenson R J, et al., J ClinInvest, 1988, 81: 951-5; Broxmeyer H E, et al., Proc Natl Acad Sci USA,1989, 86:3828-32). TRECs do not duplicate when the T-cell divides (BlomB., et al., J Immunol, 1997, 158:3571-7). As a consequence, TREC levelsare highest in recent thymic emigrants but are sequentially dilutedamongst the emigrants' progeny. TCRδ TRECs are detectable by PCR—anassay that has been shown to be a reliable tool for monitoring de novoT-cell generation (Tjormford G E, et al., J Exp Med, 1993, 177:1531-9).Absolute numbers of TREC positive cells will vary according to the totalnumber of cells analyzed. We determined that the significance of TRECpositivity would be most fairly interpreted by calculating the ratio ofthe number of TREC copies detected to the number of β-actin copiesdetected. We compared the level of TREC detected in T-cells harvestedfrom the co-cultures after 14 days to TREC levels in peripheral bloodmononuclear cells, B cells, AC133⁺ cells from the input population, andhuman fetal thymocytes. The highest TREC:bactin ratio was found inT-cells generated from the co-cultures after 14 days (0.54), followed bythymocytes from 16-22 week human fetuses (0.017). The TREC:bactin ratiosfrom fetal and adult PBMCs and from AC133⁺ bone marrow mononuclear cellswas significantly lower. These data are summarized in Table 6, below. NoTREC was detected in any of the samples of B-cells tested (n=6).

TABLE 6 Source n TREC/Bactin ratio (mean) Murine Thymocytes 6 0 BoneMarrow AC133+ progenitors 3 0.000014 Adult Peripheral blood MNCs 30.00141 Fetal Peripheral blood MNCs 1 0.0024 Fetal Thymocytes 2 0.017T-Cells generated in vitro 2 0.54

These data conclusively demonstrate that rearrangement of TCR occursduring the course of the culture period. The abundance of TREC positivecells compares favorably with that seen from fresh fetal thymus andsupports the physiologic equivalence of the in vitro system in thisaspect of T-cell differentiation.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety. What is claimed is presented below, followed by a SequenceListing.

58 1 9 PRT Artificial Sequence Homo Sapiens source 1 Glu Ala Asp Pro ThrGly His Ser Tyr 1 5 2 9 PRT Artificial Sequence Homo Sapiens source 2Ser Ala Tyr Gly Glu Pro Arg Lys Leu 1 5 3 9 PRT Artificial Sequence HomoSapiens source 3 Glu Val Asp Pro Ile Gly His Leu Tyr 1 5 4 9 PRTArtificial Sequence Homo Sapiens source 4 Phe Leu Trp Gly Pro Arg AlaLeu Val 1 5 5 10 PRT Artificial Sequence Homo Sapiens source 5 Met GluVal Asp Pro Ile Gly His Leu Tyr 1 5 10 6 12 PRT Artificial Sequence HomoSapiens source 6 Lys Ile Ser Gly Gly Pro Arg Ile Ser Tyr Pro Leu 1 5 107 9 PRT Artificial Sequence Homo Sapiens source 7 Ala Leu Ser Arg LysVal Ala Glu Leu 1 5 8 9 PRT Artificial Sequence Homo Sapiens source 8Ala Ala Arg Ala Val Phe Leu Ala Leu 1 5 9 8 PRT Artificial Sequence HomoSapiens source 9 Tyr Arg Pro Arg Pro Arg Arg Tyr 1 5 10 10 PRTArtificial Sequence Homo Sapiens source 10 Ser Pro Ser Ser Asn Arg IleArg Asn Thr 1 5 10 11 9 PRT Artificial Sequence Homo Sapiens source 11Val Leu Pro Asp Val Phe Ile Arg Cys 1 5 12 9 PRT Artificial SequenceHomo Sapiens source 12 Glu Glu Lys Leu Ile Val Val Leu Phe 1 5 13 9 PRTArtificial Sequence Homo Sapiens source 13 Glu Glu Lys Leu Ser Val ValLeu Phe 1 5 14 10 PRT Artificial Sequence Homo Sapiens source 14 Ala CysAsp Pro His Ser Gly His Phe Val 1 5 10 15 10 PRT Artificial SequenceHomo Sapiens source 15 Ala Arg Asp Pro His Ser Gly His Phe Val 1 5 10 169 PRT Artificial Sequence Homo Sapiens source 16 Ser Tyr Leu Asp Ser GlyIle His Phe 1 5 17 9 PRT Artificial Sequence Homo Sapiens source 17 SerTyr Leu Asp Ser Gly Ile His Ser 1 5 18 9 PRT Artificial Sequence HomoSapiens source 18 Met Leu Leu Ala Val Leu Tyr Cys Leu 1 5 19 9 PRTArtificial Sequence Homo Sapiens source 19 Tyr Met Asn Gly Thr Met SerGln Val 1 5 20 9 PRT Artificial Sequence Homo Sapiens source 20 Tyr MetAsp Gly Thr Met Ser Gln Val 1 5 21 9 PRT Artificial Sequence HomoSapiens source 21 Ala Phe Leu Pro Trp His Arg Leu Phe 1 5 22 9 PRTArtificial Sequence Homo Sapiens source 22 Ser Glu Ile Trp Arg Asp IleAsp Phe 1 5 23 9 PRT Artificial Sequence Homo Sapiens source 23 Tyr GluIle Trp Arg Asp Ile Asp Phe 1 5 24 15 PRT Artificial Sequence HomoSapiens source 24 Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro GlnPhe Pro 1 5 10 15 25 15 PRT Artificial Sequence Homo Sapiens source 25Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp 1 5 10 15 269 PRT Artificial Sequence Homo Sapiens source 26 Ile Leu Thr Val Ile LeuGly Val Leu 1 5 27 9 PRT Artificial Sequence Homo Sapiens source 27 LysThr Trp Gly Gln Tyr Trp Gln Val 1 5 28 9 PRT Artificial Sequence HomoSapiens source 28 Ile Thr Asp Gln Val Pro Phe Ser Val 1 5 29 9 PRTArtificial Sequence Homo Sapiens source 29 Tyr Leu Glu Pro Gly Pro ValThr Ala 1 5 30 10 PRT Artificial Sequence Homo Sapiens source 30 Leu LeuAsp Gly Thr Ala Thr Leu Arg Leu 1 5 10 31 10 PRT Artificial SequenceHomo Sapiens source 31 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val 1 5 10 329 PRT Artificial Sequence Homo Sapiens source 32 Leu Tyr Val Asp Ser LeuPhe Phe Leu 1 5 33 11 PRT Artificial Sequence Homo Sapiens source 33 SerLeu Leu Met Trp Ile Thr Gln Cys Phe Leu 1 5 10 34 9 PRT ArtificialSequence Homo Sapiens source 34 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 535 9 PRT Artificial Sequence Homo Sapiens source 35 Gln Leu Ser Leu LeuMet Trp Ile Thr 1 5 36 18 PRT Artificial Sequence Homo Sapiens source 36His Leu Tyr Gln Gly Cys Gln Val Val Pro Leu Thr Ser Ile Ile Ser 1 5 1015 Ala Val 37 9 PRT Artificial Sequence Homo Sapiens source 37 Leu LeuGly Arg Asn Ser Phe Glu Val 1 5 38 15 PRT Artificial Sequence Rubellasource 38 Trp Val Thr Pro Val Ile Gly Ser Gln Ala Arg Lys Cys Gly Leu 15 10 15 39 8 PRT Artificial Sequence Rubella source 39 Arg Val Ile AspPro Ala Ala Gln 1 5 40 15 PRT Artificial Sequence Measles source 40 HisGln Ala Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu Leu 1 5 10 15 41 9PRT Artificial Sequence Papilloma source 41 Arg Leu Cys Val Gln Ser ThrHis Val 1 5 42 9 PRT Artificial Sequence Papilloma source 42 Tyr Val ArgAsp Gly Asn Pro Tyr Ala 1 5 43 10 PRT Artificial Sequence Papillomasource 43 Gly Tyr Asn Lys Pro Leu Cys Asp Leu Leu 1 5 10 44 12 PRTArtificial Sequence Influenza source 44 Lys Gly Ile Leu Gly Phe Val PheThr Leu Thr Val 1 5 10 45 13 PRT Artificial Sequence Influenza source 45Glu Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr 1 5 10 46 9 PRTArtificial Sequence Hepatitis B source 46 Trp Leu Ser Leu Leu Val ProPhe Val 1 5 47 9 PRT Artificial Sequence Hepatitis B source 47 Phe LeuGly Gly Thr Thr Val Cys Leu 1 5 48 9 PRT Artificial Sequence Hepatitis Csource 48 Tyr Leu Val Ala Tyr Gln Ala Thr Val 1 5 49 9 PRT ArtificialSequence Hepatitis C source 49 Gly Leu Arg Asp Leu Ala Val Ala Val 1 550 14 PRT Artificial Sequence Hepatitis C source 50 Gly Tyr Lys Val LeuVal Leu Asn Pro Ser Val Ala Ala Thr 1 5 10 51 10 PRT Artificial SequenceHepatitis C source 51 Lys Leu Val Ala Leu Gly Ile Asn Ala Val 1 5 10 5215 PRT Artificial Sequence Tetanus source 52 Gln Tyr Ile Lys Ala Asn SerLys Phe Ile Gly Ile Tyr Gln Leu 1 5 10 15 53 15 PRT Artificial SequenceHomo Sapiens source 53 Thr Tyr Glu Leu Ala Pro Val Phe Val Leu Leu GluTyr Val Thr 1 5 10 15 54 15 PRT Artificial Sequence Homo Sapiens source54 Leu Lys Lys Met Arg Phe Ile Ile Gly Trp Pro Gly Gly Ser Gly 1 5 10 1555 15 PRT Artificial Sequence Homo Sapiens source 55 Lys Lys Gly Ala AlaAla Ile Gly Ile Gly Thr Asp Ser Val Ile 1 5 10 15 56 14 PRT ArtificialSequence Homo Sapiens source 56 Pro Leu Cys Ser Ala Leu Leu Val Arg GluGlu Gly Leu Met 1 5 10 57 15 PRT Artificial Sequence Homo Sapiens source57 Trp Leu Met Trp Arg Ala Lys Gly Thr Thr Gly Phe Glu Ala His 1 5 10 1558 16 PRT Artificial Sequence Homo Sapiens source 58 Val Ile Val Met LeuThr Pro Leu Val Glu Asp Gly Val Lys Gln Cys 1 5 10 15

We claim:
 1. A method for in vitro production of lymphoidtissue-specific cells, comprising: introducing an amount ofhematopoietic progenitor cells and an amount of lymphoreticular stromalcells into a porous, solid matrix having interconnected pores of a poresize sufficient to permit the hematopoietic progenitor cells and thelymphoreticular stromal cells to grow throughout the matrix, wherein thelymphoreticular stromal cells are derived from at least one lymphoidsoft tissue selected from the group consisting of spleen, liver, lymphnode, skin, tonsil and Peyer's patches, and combinations thereof, andthe amount of the lymphoreticular stromal cells is sufficient to supportthe growth and differentiation of the hematopoietic progenitor cells,and co-culturing the hematopoietic progenitor cells and thelymphoreticular stromal cells.
 2. The method of claim 1, wherein theco-culturing occurs under conditions sufficient to produce at least a10-fold increase in the number of lymphoid tissue-specific cells.
 3. Themethod of claim 1, wherein the co-culturing occurs under conditionssufficient to produce at least a 20-fold increase in the number oflymphoid tissue-specific cells.
 4. The method of claim 1, wherein theco-culturing occurs under conditions sufficient to produce at least a50-fold increase in the number of lymphoid tissue-specific cells.
 5. Themethod of claim 1, wherein the co-culturing occurs under conditionssufficient to produce at least a 100-fold increase in the number oflymphoid tissue-specific cells.
 6. The method of claim 1, wherein theco-culturing occurs under conditions sufficient to produce at least a200-fold increase in the number of lymphoid tissue-specific cells. 7.The method of claim 1, wherein the co-culturing occurs under conditionssufficient to produce at least a 400-fold increase in the number oflymphoid tissue-specific cells.
 8. The method of claim 1, wherein thehematopoietic progenitor cells are selected from the group consisting ofpluripotent stem cells, multipotent progenitor cells and progenitorcells committed to specific hematopoietic lineages.
 9. The method ofclaim 1, wherein the hematopoietic progenitor cells are derived fromtissue selected from the group consisting of bone marrow, peripheralblood, umbilical cord blood, placental blood, lymphoid soft tissue,fetal liver, embryonic cells and aortal-gonadal-mesonephros derivedcells.
 10. The method of claim 1, wherein the hematopoietic progenitorcells and the lymphoreticular stromal cells are autologous.
 11. Themethod of claim 1, wherein the hematopoietic progenitor cells and thelymphoreticular stromal cells are non-autologous.
 12. The method ofclaim 1, wherein the lymphoid tissue-specific cells are to be used intransplantation into a host and wherein the hematopoietic progenitorcells are non-autologous to the cells of the host.
 13. The method ofclaim 1, wherein the lymphoid tissue-specific cells are to be used intransplantation into a host and wherein the hematopoietic progenitorcells are autologous to the cells of the host.
 14. The method of claim1, wherein the lymphoid tissue-specific cells are to be used intransplantation into a host and wherein the lymphoreticular stromalcells are non-autologous to the cells of the host.
 15. The method ofclaim 1, wherein the lymphoid tissue-specific cells are to be used intransplantation into a host and wherein the lymphoreticular stromalcells are autologous to the cells of the host.
 16. The method of claim1, wherein the hematopoietic progenitor cells are genetically alteredhematopoietic progenitor cells.
 17. The method of claim 1, wherein thelymphoreticular stromal cells are genetically altered lymphoreticularstromal cells.
 18. The method of claims of claim 1, wherein wherein thelymphoreticular stromal cells are seeded prior to inoculating thehematopoietic progenitor cells.
 19. The method of claim 1, wherein thelymphoreticular stromal cells are seeded at the same time as thehematopoietic progenitor cells.
 20. The method of claim 1, wherein thehematopoietic progenitor cells are of human origin and thelymphoreticular stromal cells are of human origin.
 21. The method ofclaim 1, wherein the hematopoietic progenitor cells are of human originand the lymphoreticular stromal cells are of nonhuman origin.
 22. Themethod of claim 1, Wherein the porous solid matrix is an open cellporous matrix having a percent open space of at least 75%.
 23. Themethod of claim 1, wherein the porous, solid matrix having seededhematopoietic progenitor cells and their progeny, and lymphoreticularstromal cells, is impregnated with a gelatinous agent that occupiespores of the matrix.
 24. The method of claim 1, wherein the metal istantalum.
 25. The method of claim 1, wherein the hematopoieticprogenitor cells and the lymphoreticular stromal cells are cultured inan environment that is free of stromal cell conditioned medium andexogenously added hematopoietic growth factors that promotehematopoietic cell maintenance, expansion, and differentiation, otherthan serum.
 26. The method of claim 1, wherein the hematopoieticprogenitor cells and the lymphoreticular stromal cells are cultured withan exogenously added agent selected from the group consisting of stromalcell conditioned medium, and a hematopoietic growth factor that promoteshematopoietic cell maintenance, expansion, differentiation, andinfluences cell localization.
 27. The method of claim 1, furthercomprising after the co-culturing step, harvesting the lymphoidtissue-specific cells.
 28. The method of claim 8, wherein the progenitorcells committed to specific hematopoietic lineages are committed to alineage selected from the group consisting of T cell lineage, B celllineage, dendritic cell lineage, Langerhans cell lineage and lymphoidtissue-specific macrophage cell lineage.
 29. The method of claim 28,wherein the lymphoreticular stromal cells are skin stromal cells and theprogenitor cells committed to specific hematopoietic lineages arecommitted to a T cell lineage.
 30. The method of claim 9, wherein thelymphoid soft tissue is selected from the group consisting of thymus,spleen, liver, lymph node, skin, tonsil and Peyer's patches.
 31. Themethod of claim 21, wherein the nonhuman origin lymphoreticular stromalcells are of murine origin.
 32. The method of claim 22, wherein theporous solid matrix has pores defined by interconnecting ligamentshaving a diameter at midpoint, on average, of less than 150 μm.
 33. Themethod of claim 22, wherein the porous solid matrix is a metal-coatedreticulated open cell foam of carbon containing material.
 34. The methodof claim 33, wherein the metal is selected from the group consisting oftantalum, titanium, platinum, niobium, hafnium, tungsten, andcombinations thereof, wherein said metal is coated with a biologicalagent selected from the group consisting of collagens, fibronectins,laminins, integrins, angiogenic factors, anti-inflammatory factors,glycosaminoglycans, vitrogen, antibodies and fragments thereof, andcombinations thereof.
 35. The method of claim 25, wherein thehematopoietic growth factors that promote hematopoietic cellmaintenance, expansion, and differentiation, are agents selected fromthe group consisting of interleukins 3, 6 and
 11. 36. The method ofclaim 26, wherein the hematopoietic growth factor that promoteshematopoietic cell maintenance, expansion, differentiation, andinfluences cell localization, is an agent selected from the groupconsisting of interleukin 3, interleukin 6, interleukin 7, interleukin11, interleukin 12, stem cell factor, FLK-2 ligand, FLT-2 ligand, Epo,Tpo, GMCSF, GCSF, Oncostatin M, and MCSF.